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|Publication number
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|Application number
|Publication date
|Jan 28, 1992
|Filing date
|May 10, 1990
|Priority date
|Apr 23, 1987
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|Edith Wong, Michael L. Bittner
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|Monsanto Company
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|Patent Citations (6), Non-Patent Citations (26), Referenced by (16), Classifications (24), Legal Events (4)
|External Links: USPTO, USPTO Assignment, Espacenet
This is a continuation of application Ser. No. 07/041,896, filed on Apr. 23, 1987, now abandoned.
The present invention is directed to methods and compositions for producing insulin-like growth factor (IGF) in Gram-negative bacteria. Such methods and compositions provide for the secretion of IGF by recombinant Gram-negative bacterial cells. In one preferred aspect, the IGF is secreted into the periplasmic space as a mature protein.
Insulin-like growth factors (IGF's) have been isolated from various animal species and are believed to be active growth promoting molecules that mediate the anabolic effects of such hormones as growth hormone and placental lactogen. To date, several classes of IGF's have been identified. These include insulin-like growth factor-I (somatomedin C), insulin-like growth factor-II, Somatomedin A and a mixture of peptides called "multiplication-stimulating activity." This heterologous group of peptides exhibit important growth-promoting effects in vitro (Daughday, 1977; Clemmons and Van Wyk, 1981) and in vivo (Van Baul-Offers and Van de Brande, 1980; Schoenle, 1982).
Insulin-like growth factor-I (IGF-I) is a 70 amino acid basic protein (Rinderkneckt and Humbel, 1978; Rubin et al., 1982) which has been demonstrated to play a fundamental role in postnatal mammalian growth as a major mediator of growth hormone action (Copeland et al., 1980; Zapf et al., 1981 and Schoenle et al., 1982). As such, IGF-I is useful in the treatment and/or potentiation of various growth related conditions.
The ability to obtain large quantities of IGF-I for commercial and therapeutic purposes has been hampered by the fact that the content of IGF-I in mammalian tissues is very low and, in contrast to insulin-like growth factor-II, no cultured mammalian cell lines have as yet been identified which elaborate significant quantities of IGF-I.
Natural sources of IGF-I thus far having proven inadequate to supply research and clinical needs, individuals and groups have turned to recombinant DNA techniques for the manufacture of IGF-I. Examples of such recombinant DNA methodologies can be found in European Patent Publication No. 155,655 (published Sept. 25, 1985) which described production of IGF-I as a cytoplasmic fusion protein; 135,094 (published Mar. 12, 1085) which describes a process for producing IGF-I as a fusion protein in E. coli; 130,166 (published Jan. 2, 1985) which describes production of IGF-I as a cytoplasmic fusion protein in Staphylococcus aureus and production as a cytoplasmic protein containing an amino-terminal methionine in E. coli 128,733 (published Dec. 19, 1984) which describes a process for producing IGF-I in the yeast Saccaromyces cerevisiae employing a yeast α-factor, a-factor or acid phosphatase signal sequence, and describes production of IGF-I as a cytoplasmic fusion protein; Nilsson, B. et al. (1985) which describes the cytoplasmic production of IGF-I as a Staphylococcal protein A - IGF-I fusion protein; and Buell et al. (1985) which describes intracellular production of IGF-I as a mature and fusion protein.
A process for producing IGF-I as a secreted protein in Gram-negative bacteria such as E. coli has not yet been disclosed. Such a process is desirable as current methods for production of IGF-I employing cytoplasmic production have required the use of fusion protein constructs to protect the mature IGF-I protein product from degradation by cytoplasmic proteases. (see European Pat. application publication No. 135,094 supra and Buell et al. 1985). Fusion protein constructs have the disadvantage of requiring an additional production step consisting of release of mature IGF from the fusion protein. Such release is typically mediated by enzymatic or chemical means, which means can be harmful to the desired IGF-I protein and/or can require modifications in IGF-I protein structure (see e.g. European Pat. application publication No. 135,094 supra). Secretion of mature IGF-I into the periplasm of such Gram-negative bacteria as E. coli would afford a compartmentalization of the IGF-I between the inner and outer cell membranes of the host cells thereby protecting the IGF-I from harmful cytoplasmic enzymes. Additionally, secretion into the periplasmic space allows for production of the IGF-I as a mature protein thereby eliminating the cumbersome and often costly release of mature protein from a fusion construct or otherwise undesirable amino terminal amino acids. Furthermore, production of IGF's in E. coli avoids the possibility of undesirable glycosylation which may occur when yeast host cells are employed.
Examples of various secretion systems described for use in E. coli include U.S. Pat. No. 4,336,336 (filed Jan. 12, 1979); European Pat. application Publication Nos. 184,169 (published June 11, 1986), 177,343 (published Apr. 9, 1986) and 121,352 (published Oct. 10, 1984); Oka, T. et al. (1985); Gray, G. L. et al. (1985); Ghrayeb, J. et al. (1984) and Silhavy, T. et al. (1983). Briefly, these systems make use of the finding that a short (15-30) amino acid sequence present at the amino (NH2 -terminus of certain bacterial proteins, which proteins are normally exported by cells to noncytoplasmic locations, are useful in similarly exporting heterologous proteins to noncytoplasmic locations. These short amino acid sequences are commonly referred to as "signal sequences" as they signal the transport of proteins from the cytoplasm to noncytoplasmic locations. In Gram-negative bacteria, such noncytoplasmic locations include the inner membrane, periplasmic space, cell wall and outer membrane. At some point just prior to or during transport of proteins out of the cytoplasm, the signal sequence is typically removed by peptide cleavage thereby leaving a mature protein at the desired noncytoplasmic location. Site-specific removal of the signal sequence, also referred to herein as accurate processing of the signal sequence, is a preferred event if the correct protein is to be delivered to the desired noncytoplasmic location.
Attempts to employ such previously described signal sequences and secretion systems for delivery of heterologous proteins into the E. coli periplasmic space have, however, not always been successful. (see Kadonaga, J. et al., 1984; Ohsue, K. et al., 1983). Such results indicate that further studies are needed to clarify the secretion mechanism before secretion of heterologous proteins in such Gram-negative bacteria as E. coli can become a predictable phenomenon. The term "heterologous protein" is herein understood to mean a protein at least a portion of which is not normally encoded within the chromosomal DNA of a given host cell. Examples of heterologous proteins include hybrid or fusion proteins comprising a bacterial portion and a eucaryotic portion, eucaryotic proteins being produced in procaryotic hosts, and the like.
The present invention is directed to methods and compositions for producing insulin-like growth factor-I (IGF-I) in Gram-negative bacteria whereby the IGF-I so produced is secreted into the periplasmic space of the bacteria.
In one embodiment of the present invention, the methods comprise causing expression of a gene in Gram-negative bacteria, wherein the gene comprises a DNA sequence encoding a major outer membrane protein signal sequence operatively joined to a DNA sequence encoding IGF-I, so that the IGF-I produced within the bacteria is secreted into the periplasmic space thereof. It has been discovered that the lamB and ompF signal sequences provide for a site-specific release of the signal sequence from the IGF-I protein so that the IGF-I protein can be released into and accumulate at relatively high levels in the periplasmic space of selected bacteria.
Also provided are various synthetic DNA sequences and transformed bacteria useful in practicing the novel methods as provided herein.
In the following diagrammatic representation, the directional arrows represent the 5' to 3' orientation of DNA coding sequence(s) (e.g. direction of transcription of a DNA coding sequence), "amp4 " denotes an ampicillin resistance gene, "P" denotes a promoter sequence operable in E. coli, "R.B.S." denotes a ribosome binding site, "LAM" denotes a DNA sequence coding for a lamB signal sequence, "IGF-Iy " denotes an IGF-I coding sequence comprising yeast preferred codons, "IGF-IE " denotes a IGF-I coding sequence comprising E. coli preferred codons and "T" denotes a transcription termination sequence. Relevant restriction endonuclease sites are also shown. The DNA regions marked as described above are for diagrammatic purposes only and are not drawn to scale unless otherwise noted.
FIG. 1 depicts the construction of a synthetic double-stranded (ds) IGF-Iy DNA coding sequence. The four chemically synthesized oligonucleotide portions of the synthetic coding sequence are shown in capital letters and are numbered IGF-1 (1) (2) (3) & (4). Regions of overlap between the chemically synthesized oligomers are denoted by a solid line (--) between complementay nucleotides. The lower case letters denote enzymatically generated oligonucleotides. The amino acid sequence of the IGF-I peptide encoded in the synthetic ds DNA sequence is shown in an amino (NH2)-terminal to carboxyl (COOH)-terminal orientation.
FIG. 2 depicts the construction of pMON2108, and IGF-I expression vector comprising a DNA sequence coding for a lamB signal sequence operatively joined to an IGF-I coding sequence consisting of E. coli preferred codons.
FIG. 3 A & B depict the construction of pMON2123, and IGF-I expression vector comprising a DNA sequence coding for an ompF signal sequence operatively joined to an IGF-I coding sequence consisting of E. coli preferred codons. "ompF" denotes the DNA coding sequence for the ompF signal sequence.
As used herein, the symbols representing amino acids (i.e. Ala for alanine) and nucleotides (e.g. C,G,A,T,) are those conventionally employed (Lehninger, 1976).
The present invention provides a means for producing IGF-I in Gram-negative bacteria whereby the IGF-I so produced is secreted into the periplasmic space of the bacteria. The discovery of such a means is significant as it allows for the direct production of mature IGF-I free from any undesirable amino acids and allows for an accumulation of mature protein in a readily accessible site and at a site free from harmful cytoplasmic proteases. The term "mature" when used to characterize and IGF-I protein means a protein essentially free from any extraneous NH2 - or carboxy(COOH)-terminal amino acids. An example of such a protein is one having the primary amino acid sequence as shown in FIG. 1. Furthermore, the processes and compositions of the present invention provide for production of IGF-I at relatively high levels (e.g. 4 to 8% of total cellular protein produced).
The signal sequences which were discovered to effect high level production and secretion of accurately processed IGF-I are two signal sequences of major E. coli outer membrane proteins. E. coli possess a plurality of major outer membrane proteins including, but not limited to, lamB, ompF, ompA, ompC and phoE. These proteins are all synthesized in the cytoplasm and transported to their outer membrane location. As such, these proteins all contain a signal sequence which is processed either before or during transport of the mature outer membrane protein from the cytoplasm to its noncytoplasmic location. As described more fully hereinafter, the signal sequences of the E. coli lamB and ompF proteins provide accurate processing of the signal sequence when operatively joined to IGF-I peptides. The lamB and ompF signal sequences also provide for secretion and accumulation of mature IGF-I in the periplasmic space of the Gram-negative (e.g. E. coli) host cells and allow high level production of IGF-I by the transformed host. Based upon these findings, it is believed that other E. coli major outer membrane protein signal sequences can effect the secretion and accumulation of mature IGF-I in the periplasmic space.
It is known that all Gram-negative bacteria possesses an outer membrane. It is also known that most members of the family Enterobacteriaceae possess a maltose-inducible outer membrane protein homologous to the lamB protein of E. coli (see Bloch and Desaymard, 1985, incorporated herein by reference hereto). Thus, it is expected that at least some of the lamB signal sequences of other Enterobacteriaceae, such as Shigella, Enterobacter, and Klebsiella, can effect secretion of mature IGF-I into the periplasmic space particularly when the host cell belongs to the same or sufficiently homologous genus and species from which the outer membrane protein signal sequence was derived.
In one embodiment of the present invention employing an E. coli expression system described more fully hereinafter, the preferred lamB signal sequence comprised the following amino acid sequence: ##STR1## and the preferred ompF signal sequence comprised the following amino acid sequence: ##STR2##
Significantly, several other E. coli non-outer membrane protein signal sequences reported to effectively secrete E. coli and/or heterologous proteins into the periplasmic space were found to be unable to secrete accurately processed mature IGF-I into the periplasmic space and/or did so at levels significantly lower than the lamB or ompF systems. The failure of such other signal sequences to effect secretion of mature IGF-I into the periplasmic space to a significant degree is in keeping with prior reports of inabilities to predict the secretion efficacy of a given signal sequence for a given peptide.
In order to effect secretion of mature IGF-I using the lamB or ompF signal sequences, the signal sequences are operatively joined to the IGF-I peptide. For the lamB signal, such operative joining can be achieved by at least two distinct means. In one preferred means, the lamB signal sequence was directly joined (e.g. by peptide bonding) at its carboxyl-terminus to the amino-terminus of the IGF-I peptide. The lamB signal sequence and IGF-I peptide are thus contiguous with one another. Alternatively, the lamB signal sequence is operatively joined to the IGF-I peptide by creating a hybrid (heterologous) protein containing sequentially from the NH2 -terminus a lamB signal sequence, at least 39 to 43 of the NH2 -terminal amino acids of the E. coli lamB protein and an IGF-I peptide (see Silhavy, et al., 1977; Silhavy et al., 1976). The DNA sequence of the E. coli lamB protein has been published by Clement and Hofnung (1981) which publication is hereby incorporated herein by reference hereto. In a preferred embodiment, the NH2 -terminal portion of the mature lamB protein comprised the following amino acid sequence; ##STR3## In both instances, when employing the expression vectors and systems hereinafter described, mature IGF-I is successfully secreted into the periplasmic space of the Gram-negative host cell and is produced at high levels (e.g. 4-8% of total cellular protein produced).
In one embodiment of the present invention, operative joining of an ompF signal sequence to an IGF-I peptide is achieved by similarly constructing (e.g. producing) heterologous proteins consisting of an ompF signal sequence at the NH2 -terminus and an IGF-I peptide at the COOH-terminus so that the signal sequence and IGF-I peptide are contiguous with one another. These heterologous proteins successfully secreted mature IGF-I into the periplasmic space of the Gram-negative host cells. Furthermore, the IGF-I so secreted is accumulated at levels similar to those achieved using a lamB signal sequence.
Having discovered that such major outer membrane protein signal sequences as the lamB and ompF signal sequences effect the secretion of mature IGF-I into the periplasmic space of such Gram-negative bacteria as E. coli, it is now possible to use conventional recombinant DNA techniques to produce the heterologous proteins described above (e.g. proteins consisting of a lamB or ompF signal sequence operatively joined to an IGF-I peptide).
In one embodiment of the present invention, production of heterologous proteins comprising a lamB or ompF signal sequence operatively joined to an IGF-I peptide is achieved as follows. A synthetic gene is constructed which contained the following components sequentially aligned in a 5' to 3' orientation: a promoter, a ribosome binding site, a translation start/signal codon contiguous with a DNA sequence encoding the heterologous protein sought to be produced and a translation stop codon. Such a synthetic gene can be constructed by conventional recombinant DNA techniques (see Maniatis et al., 1982) and by employing the methods and compositions set forth herein. The term "gene" refers to DNA sequences which provide for production of protein(s) encoded therein. The term "synthetic" is understood to mean a chemically and/or enzymatically generated nucleic acid sequence or molecule free from naturally occurring intervening or contiguous sequences. Also embraced in the term "synthetic" are chemically and/or enzymatically generated molecules or sequences which are combined with nucleic acid sequences or molecules with which they are not naturally found associated or combined. Examples of synthetic genes, molecules or sequences include, but are not limited to, nucleic acid sequences enzymatically or chemically released from a larger (e.g. naturally occurring) molecule and genomic DNA which has been modified by nucleic acid addition, deletion and/or rearrangement. The term "genome" or "genomic" as used herein means the total DNA (e.g. chromosomal and extrachromosomal) contained within a given cell.
In constructing a synthetic gene or genes which encode a heterologous protein comprising a lamB or ormpF signal sequence operatively joined to an IGF-I peptide, any promoter capable of causing transcription in Gram-negative bacteria of such a gene or genes can be employed. Examples of such promoters are well known and available to those skilled in the art and include, without limitation, bacterial, viral and plasmid gene promoters (Rosenberg and Court, 1979). DNA molecules containing such promoter sequences can be isolated either by cloning the desired regions or, more conveniently, by chemically synthesizing them. A preferred promoter is the E. coli recA promoter, the sequence of which has been published (see Sancar & Rupp, 1979; and Sancar et al. 1980) and is furthermore contained on plasmid pMON2690 as a BamHI restriction fragment (see FIG. 2). E. coli carrying plasmid pMON2690 have been deposited with the Americal Type Culture Collection (Rockville, Maryland) under accession number 67325.
Similarly, DNA sequences encoding the desired heterologous proteins which proteins can effect the secretion of IGF-I and preferably, mature IGF-I peptides into the periplasmic space, can also be chemically or enzymatically synthesized. The sequences of the lamB and ompF signal sequences have been published (Silhavy et al., 1983; Nagahari et al., 1985) as have the sequences of the lamB and ompF proteins (Inokuchi, K. et al., 1982, 1984; Silhavy, T. et al., 1983). Thus, DNA sequences encoding these proteins or any desired fragment thereof can be synthesized or isolated from appropriate gene or cDNA libraries by conventional methods. Thereafter, the synthetic DNA sequence encoding the desired lamB or ompF signal sequence can be operatively joined, by such means as ligase, to a DNA sequence encoding IGF-I.
The primary amino acid sequence of IGF-I has been published (Rinderknecht and Humbel, 1978) and is shown in FIG. 1. From the primary amino acid sequence, a plurality of DNA coding sequences can be constructed based upon the genetic code. Selection of the specific amino acid codons to be employed in generating an IGF-I DNA coding sequence is generally guided by such factors as determining a sequence which will optimize transcription, translation and, ultimately, the amount of protein produced. Selection of codons is thus, for example, guided by a desire to avoid potential secondary structure constraints, a preferred use of codons by the host cell (Fiers et al., 1976; Maruyama, T. et al., 1986; Sharp, P. et al., 1986; Bennetzen, J. and Hall, B., 1982; Gouy, M. and Gautier, C., 1982) and/or the need to provide for desired restriction endonuclease cleavage sites. An example of an IGF-I DNA coding sequence consisting of yeast preferred codons is shown in FIG. 1. An exemplary method for its construction is detailed in the examples below. Alternatively, an IGF-I coding sequence can be isolated from an appropriate cDNA library by conventional means.
As previously indicated, the synthetic genes of the present invention contained a ribosome binding site. In prokaryotic systems, a ribosome binding site such as a Shine-Dalgarno sequence (Shine and Dalgarno, 1974) has been demonstrated to be required for efficient translation of messenger RNA molecules. Recently, a novel ribosome binding site containing sequences from the gene 10 of bacteriophage T7 has been shown to be particularly effective in enhancing protein production. These novel bacteriophage T7 gene 10 sequences, herein referred to as "G10L sequences," are described more fully in U.S. Pat. application Ser. No. 005821 filed on Feb. 4, 1987 now abandoned which is incorporated herein by reference hereto. The preferred synthetic genes of the present invention contained G10L sequences operatively joined to DNA sequences coding for the heterologous proteins of the present invention (e.g. proteins comprising a lamB or ompF signal sequence operatively joined to an IGF-I peptide).
In another preferred embodiment, the synthetic genes of the present invention further contain transcription termination sequences. Transcription termination sequences useful in the present invention include, without limitation, those found in bacteriophage and Gram-negative bacterial DNA (Holmes et al., 1983). A preferred transcription termination sequence derived from the gene 10 of bacteriophage T7 (Dunn and Studier, 1983(is described in U.S. Pat. application Ser. No. 005821 (supra).
Expression of the synthetic genes of the present invention and bacterial production of the heterologous proteins encoded therein is achieved as follows. The synthetic genes are inserted into expression vectors which vectors are then employed to transform such Gram-negative bacterial host cells as E. coli and Pseudomonas. Suitable host cells are identified by their respective abilities to provide for transport of IGF-I to the periplasm and accurate processing (e.g. removal) of the signal sequence in accordance with procedures described herein. An expression vector or vehicle is herein understood to comprise a DNA molecule such as a phage, plasmid or cosmid DNA capable of transforming a host cell, able to replicate genes inserted therein and facilitate expression of such genes in the transformed host cell. The term "transform" or "transformation" is herein understood to comprise any method for introducing exogenous DNA into the genome of a host cell. Such methods include, without limitation, transfection, transduction, infection and integration into chromosomal DNA. Any expression vector such as phage, plasmid or cosmid vectors known and/or available to those skilled in the art can be employed (see e.g. Maniatis et al., 1982). The preferred vectors included such pBR plasmid vectors as pBR327 (Soberon et al., 1980).
The insertion of the synthetic genes into expression vectors and/or construction or assembly of such genes therein can employ conventional methods known to those skilled in the art for insertion into or joining of nucleic acid molecules. Such methods include, without limitation, blunt-end or sticky-end ligation or chemical synthesis of desired coding or functional oligonucleotide sequences. FIG. 2 presents illustrations of two preferred expression vectors (denoted pMON2690 and pMIN2108) carrying preferred synthetic genes of the present invention. These vectors contain a pBR327 origin or replication which permits autonomous replication of the vector in such host cells as E. coli and contains a drug resistance marker (ampr) allowing selection of bacterial cells carrying (e.g. transformed with) the vectors.
Once such a vector is constructed, the essential components can be readily exchanged for alternative components by conventional recombinant DNA techniques. For example, by using the indicated restriction endonuclease sites additional and/or alternative DNA sequences can be inserted into the vectors by enzymatic or chemical linkage. Indeed, by employing the restriction endonuclease sites flanking that region of the expression vector containing DNA sequences encoding the ribosome binding site and lamB signal sequence, an alternate signal sequence, such as ompF, is inserted. By analogous means, alternate promoters, ribosome binding sites, and/or markers can be inserted into the expression vector.
In one embodiment, expression vectors containing a synthetic gene encoding a heterologous protein comprising a lamB or ompF signal sequence operatively joined to an IGF-I peptide were employed to transform Gram-negative host cells such as E. coli. Preferred host cells include E. coli strain SE6004 described by Liss et al. (1985) and Emr et al. (1981) and other such Gram-negative bacteria whose signal peptidase(s) is (are) able to accurately cleave the peptide bond joining the signal sequence and the heterologous peptide. E. coli strain SE6004 has been deposited with the Americal Type Culture Collection (ATCC) under accession number 53588.
The transformed host cells are then selected (Maniatis et al., 1982) and cultured under conditions which permit or cause expression of the synthetic gene. The resultant protein produced, comprising a lamB or ompF signal sequence operatively joined to an IGF-I peptide, provides for the efficient transport and accumulation of IGF-I peptides in the periplasmic space of the transformed host cell. Furthermore, the IGF-I so produced and accumulated is produced at high levels (at least 4-8% of total cellular protein), the signal sequences are accurately processed by the host cell and is biologically active.
Periplasmic proteins can then be selectively purified by such methods as lysozyme-EDTA (ethylenediaminetetraacetic acid) treatment (Oliver and Beckwith, 1982), spheroplasting (Neu et al., 1964), freeze-thaw treatments (European Patent Publication No. 1774,343, published Apr. 9, 1986) or by osmotic shock (Oka et al., 1985). Insoluble proteins can be solubilized in such solvents as urea (e.g. 1.6 grams (wet weight) of cell pellet per milliliter of buffer comprising 2-6 M urea) preferably at an alkaline pH and in the presence of a reducing agent, such as dithiothreitol. The solubilized proteins are then refolded by oxidization.
The levels of protein (e.g. IGF-I produced) can be determined by such conventional assays as Western immunoblotting, radioimmunoassay, protein staining of a protein gel and/or enzymatic activity. Examples of assays available for determination of IGF-I levels and bioactivity include, without limitation, radioimmunoassay (Copeland et al., 1980), radioreceptor assay (D'Ercole et al., 1976) and cell growth assays (Stiles et al., 1979; Kotts, C. E., 1984). By such means, it was determined that the IGF-I produced in accordance with the methods of the present invention was biologically active and represented from about 3 to 10% of the total produced by the transformed Gram-negative bacteria.
In some instances, a fraction of the proteins secreted into the periplasmic space leak through the host cell's outer membrane into the media. Indeed, in the secretion systems described herein, about 3 to 5% of the IGF-I protein produced can be purified from the growth medium. The percentage of protein which leaks into the culture medium can be increased, if desired, by employing such methods described in U.S. Pat. No. 4,595,658 to Zinder et al. Briefly, cells able to secrete the desired protein (e.g. IGF-I) into the periplasmic space are genetically altered by insertion of the f1 bacteriophage gene III into their genomes. Production of the gene III protein by Gram-negative bacteria such as E. coli causes perturbations in the outer membrane which, in turn, allows periplasmic proteins to leak out of the cell and into the media. Retrieval of proteins from the media can have advantages in such situations as when continuous culture conditions are being employed.
The following microorganisms have been deposited with the Americal Type Culture Collection (ATCC), 12301 Parkalwn Drive, Rockville, Maryland 20852, U.S.A. in accordance with the Budapest Treaty:
ATCC 53588: E. coli SE6004
ATCC 67325: E. coli strain 294 (pMON2690)
It should be understood that the availability of a deposit does not constitute a license to practice the subject invention in derogation of patent rights granted by governmental action. Furthermore, the present invention is not to be limited in scope by the microorganisms deposited since the deposited embodiments are intended only as specific illustrations of the invention.
All oligonucleotides were synthesized employing a 380A Applied Biosystems, Inc. DNA synthesizer in accordance with the procedure set forth by the manufacture, Applied Biosystems, Inc., (Foster City, California). Unless otherwise noted all specialty chemicals were obtained from Sigma (St. Louis, Missouri). Restriction enzymes and DNA modifying enzymes were purchased from New England Biolabs (Beverly, Mass.), New England Nuclear (Boston, Mass.) or Bethesda Research Laboratories (BRL) (Gaithersburg, Maryland) and used in accordance with manufacturer's directions. T4 DNA ligase was purchased from Promega Biotec (Madison, Wisc.) and used in accordance with manufacturer's specifications. 32 P-labeled nucleotides and I125 -labeled protein A were purchased from Amersham (Arlington Heights, Ill.). E. coli strains SE2060 and SE6004 were obtained from Dr. Thomas Silhavy, Department of Molecular Biology, Princeton University, Princeton, N.J. 08544 and are described by Emr et al. (1981). E. coli strain SE6004 carrying a pr1A4 mutation can be obtained from the ATCC (Rockville, Maryland) under ATCC accession number 53588. E. coli strains W3110, 294 and MJ101 can be obtained from the ATCC (Rockville, Maryland) under ATCC accession Nos. 27325, 31446 and 33876, respectively. All bacterial growth media components and antibiotics were obtained from either Sigma (St. Louis, Miss.) or Difco Laboratories (Detroit, Mich.). pBR327 vectors can be obtained from Pharmacia (Piscataway, N.J.).
The growth media for the E. coli and conditions for selection of bacterial cells carrying plasmids containing an ampicillin resistance (ampr) marker were as described in Maniatis et al. (1982). When employed for protein expression, E. coli were grown in Laria Broth (LB) or M9 minimal medium (Maniatis et al., 1982) supplemented with 100 μg/ml ampicillin. Transformation of E. coli host cells with recombinant vectors was performed as described in Maniatis et al. (1982).
Induction of transcription from the recA promoter was conducted briefly as follows. Overnight cultures of E. coli host cells carrying expression plasmids were diluted to 20-25 Klett units (measured with a Klett-Summerson meter using a green filter, Klett Mfg. Co., New York, New York) in M9 minimal media supplemented with 0.25% (w/v) glucose and 1% (w/v) casamino acids and 0.25 μg/ml thiamine and grown to a cell density of 150-180 Klett units. The cells were then induced by adding nalidixic acid to the growth media at a final concentration of 50 μg/ml. Growth was continued for several hours at 37� C. with aliquots taken at 2 or 3 hours after induction for heterologous protein analysis. A high level of aeration was maintained throughout the growth in order to achieve maximal production of the desired gene product.
Periplasmic proteins produced in the E. coli host cells were obtained by an osmotic shock procedure essentially as described by Oka, T. et al. (1985). Specifically, cells from 30 ml of culture were pelleted and the supernatant collected. The supernatant was called the "media fraction." The pelleted cells were then resuspended in 5 ml of 20% (w/v) sucrose and 30 mM TRis pH 7.5. EDTA was then added to give a final EDTA concentration of 1�10-3 M. The resuspended cells were then incubated for 15 minutes at room temperature (about 20� C.) after which the cells were pelleted. The resulting supernatant was called the "sucrose fraction." The pelleted cells were resuspended in 3 ml of ice water (about 0-4� C.) and shaken gently for 10 minutes in an ice bath and then pelleted. The resulting pellet and supernatant were called the "lysate" and "water wash," respectively. Typically, periplasmic proteins obtained by an osmotic shock procedure are found in the water fresh fraction. The periplasmic IGF-I, however, was predominantly found in the sucrose fraction.
IGF-I was purified from the media as follows. Bacterial cells were removed by pelleting or passing the media through a 0.22μ membrane filter (Millipore, Inc.; Bedford, Mass.) after which the media was concentrated using a YM-3 membrane (Amacon Filter Co., Danvers, Mass.) and the protein then precipitated with 60% (w/v) ammonium sulfate. The precipitate was then pelleted, the pellet dissolved in 50 mM sodium formate (pH 4.2) and then desalted on a Sephadex G-25 column (Pharmacia, Piscataway, N.J.) after which the eluatre was subjected to cation exchange chromatography on a mono S column (Pharmacia, Piscataway, N.J.) from which the IGF-I was eluted with a 0-1 M sodium chloride gradient. The protein eluted at about 400 to 500 mM Nacl. The IGF-I containing fractions from the mono S column were then subjected to reverse phase high performance liquid chromatography on a Brownlee C8 RP300 Aquapore column (Brownlee Laboratories; Santa Clara, Calif.) from which the IGF-I was eluted with an acetonitrile-trifluoroacetic acid (TFA) gradient containing 0.1% (v/v) TFA. The IGF-I eluted at about 30 % (v/v) acetonitrile. The recovered IGF-I was then stored at -20 to -80� C. as a lyophilized powder.
The levels of IGF-I produced by the bacterial host cells were determined by protein-specific assays such as protein staining (with Coomassie Brilliant Blue R250) of 15% (w/v) sodium dodecyl sulfate (SDS)-polyacrylamide gels (Laemmli, 1070) or by Western immunoblotting as described by Renart et al. (1979). Specifically, total protein produced within the host cell (cytoplasmic and periplasmic), protein secreted into the media and protein secreted only into the periplasmic space were determined as follows. Pelleted cells from 1 ml of culture were dissolved in SDDS sample buffer as described by Laemmli (1970) to a final concentration of 1 Klett unit per μl. This sample represented the total protein produced within the host cell. The media fraction, sucrose fraction, water wash and lysate (all described above) were individually precipitated with trichloracetic acid (TCA) and then redissolved in SDS sample buffer to a final concentration of 1 Klett unit/μl. All samples in SDS sample buffer were heated at 90� C. to 100� C. for 5 minutes prior to electrophoresis in 15% (w/v) SDS polyacrylamide gels (Laemmli, 1970). The Coomassie Brilliant Blue protein-stained gels were scanned by a Joyce Loeble chromoscan 3 scanning densitometer (Joyce Loeble, Inc., New Castel, Great Britain).
IGF-I specific antibody was prepared in accordance with the method of Vaitukaitis, J. L. (1981). Briefly, female New Zealand white rabbits were immunized intradermally with about 100 μg of IGF-I in Freund's Complete Adjuvant. The rabbits received intradermal booster injections of about 100 μg of IGF-I in Freund's Complete Adjuvant at 4 and 8 weeks.
Amino acid sequence analysis of bacterially produced heterologous proteins was performed using an Applied Biosystems, Inc. (Foster City, California) Model 470A gas phase protein sequencer according to the methods described by Hunkapiller et al. (1983). The respective phenylthiodantoin (PTH)-amino acid derivatives were identified by reverse phase high performance liquid chromatography in an on-line fashion employing an Applied Biosystems, Inc. (Foster City, California) Model 120A PTH Analyzer fitted with a Brownlee 2.1 mm inner diameter PTH-C18 column.
The bioactivity of IGF-I peptides isolated from the periplasmic space of recombinant gram negative bacteria was established by the rat L6 myoblast proliferation assay described by Kotts, C.E. (1984) and U.S. Pat. application Ser. No. 777,117 now abandoned which is incorporated herein by reference hereto. Specifically, IGF-I contained within the sucrose fraction following osmotic shock isolation was compared to a commercial preparation of human IGF-F obtained from Amgen, Inc. (Thousand Oaks, Calif., Lot 407) for demonstrable physiologicla activity in that assay. In order to minimize or eliminate any effects of sucrose in the L6 myoblast proliferation assay, the sucrose fraction samples (e.g. 2 ml of each sample) were first dialyzed versus one liter 44 mM NaHCO3, pH 7.8, at 4� C., with three changes of dialysate over a three day period. The dialysis tubing had a molecular weight cut-off of 1000 kilogaltons. In all rat L6 myoblast proliferation assays conducted with the periplasmic IGF-I of the present invention, the IGF-I exhibited a statistically significant biological activity.
Radioimmunoassay analysis of both the dialyzed and undialyzed sucrose fraction was performed using a somatomedin C radioimmunoassay kit obtained from Nichols Institute (San Juan Capistrano, Calif.) in accordance with manufacturer's instructions.
This example described the construction and assembly of the synthetic double-stranded IGF-I coding sequence shown in FIG. 1. In this Example 1, the coding portion of the sequence predominantly comprises yeast preferred codons as determined from the codon usage pattern in the yeast 3-phosphoglycerate kinase gene (Genbank Data base; see Bilofsky et al., 1986). Less preferred codons were also employed at some sites in the sequence in order to create or remove restriction sites and/or to minimize the possibility of competing secondary structure within the individual oligonucleotides. Oligonucleotides were synthesized as previously described and their respective concentrations determined using quantitative 5'-end labeling with γ32 P-ATP followed by separation of unincorporated γ32 P-ATP using ascending paper cromatography.
Construction and assembly of the synthetic IGF-I coding sequence was conducted in accordance with the methods described by Scarpulla et al. (1982); Rossi et al. (1982); and Adams & Galluppi (1986). Specifically, in order to produce the synthetic dsDNA sequence shown in FIG. 1, four partially overlapping synthetic oligonucleotides, designated IGF-I (1), (2), (3) and (4) and denoted by the capital letters, were synthesized. The oligonucleotides were annealed by mixing approximately equimolar quantities of each overlapping segment ]e.g. IG-I (1) was mixed with (2) and IGF-I (3) was mixed with (4)] and heating to 100� C. for about 2 min., cooling slowly (about 20 min.) to room temperature (about 20� C.) and incubating at room temperature for about 15 min. After annealing, the individual mixtures were diluted to a final concentration of 1 μM and incubated with about 2 units of E. coli DNA polymerase, Klenow fragment, in a buffer containing 10 mM Tris pH 8.0, 10 mM MgCl2 and 0.25 mM of each deoxynucleoside triphosphate for 30 to 45 min. at room temperature. The reaction was stopped by heating to 70� C. for 10 min. and the DNA product then precipitated with ethanol. The dsDNA products which ranged from about 129 nucleotides for IGF-I (1) and (2) and about 128 nucleotides for IGF-I (3) and (4) were individually purified by electrophoresis on 8% (w/v) polyacrylamide gels containing 10% (v/v) glycerol. Multiple copies of each dsDNA product were then generated by cloning the products into a cloning vector. Useful cloning vectors include those described in Maniatis et al. (1982) and wherein the preferred cloning vectors include those vectors which facilitate DNA sequencing of the newly synthesized dsDNA product. Such preferred vectors include, but are not limited to pEMBL plasmids (Dente et al., 1983) and pTZ plasmids which contain a single-stranded origin of replication. Plasmids containing single-stranded origins of replication can be obtained from Pharmacia (Piscataway, N.J.). Once multiple copies of the dsDNA products were generated the products were excised from the cloning vectors and the IGF-I (1) and (2) dsDNA product ligated to the IGF-I (3) and (4) dsDNA product with T4 DNA ligase to form a dsDNA sequence coding for mature IGF-I.
This example describes the construction of various expression vehicles comprising synthetic IGF-I DNA coding sequences operatively joined to a secretion signal sequence. Specifically, expression vectors comprising a synthetic IGF-I DNA coding sequence consisting of either y east or E. coli preferred codons operatively joined to a lamB or ompF signal sequence are described.
a. lamB
FIG. 2 presents diagrammatic representations of two expression plasmids, pMON2690 and pMON2108, which contain a DNA sequence encoding IGF-I operatively joined to a lamB signal sequence. The two plasmids are identical except that pMON2690 contains an IGF-I coding sequence comprising yeast preferred codons (denoted IGF-Iy) and pMON2108 contains an IGF-I DNA coding sequence comprising E. coli preferred codons (denoted IGF-IE). E. coli strain 294 carrying plasmid pMON2690 has been deposited with the ATCC and given ATCC accession number 68325.
pMON2690 is a pBR327 plasmid having inserted therein the following DNA sequences in a 5' to 3' sequential orientation: a promoter (P), a ribosome binding site (R.B.S.), lamB signal sequence (LAM), IGF-I coding sequence (IGF-Iy) and a transcription termination sequence (T). Specifically, the promoter sequence comprised a recA promoter, the ribosome binding site comprised of a 100 base pair G10L sequence, the lamB signal sequence comprised a DNA sequence encoding the preferred lamB signal sequence previously described, the IGF-I coding sequence comprised the sequence shown in FIG. 1 and the transcription termination sequence comprised a bacteriophage T7 gene 10 transcription termination sequence. The DNA sequences contained within plasmid pMON2690 were either chemically and/or enzymatically synthesized, as described above, or isolated, by such recombinant DNA techniques a restriction cleavage (Maniatis et al., 1982), from available vectors containing the desired sequence(s). Once so generated, the desired DNA sequences were inserted into a pBR327 plasmid to achieve the described 5' to 3' sequence orientation.
As previously indicated, plasmid pMON2108 is identical to pMON2690 except that pMON2108 has an IGF-I DNA coding sequence comprising E. coli preferred codons (IGF-IE) in lieu of yeast preferred codons. The IGF-IE DNA coding sequence contained within plasmid pMON2108 was chemically and enzymatically constructed in a manner analogous to the method described above for construction of the IGF-I DNA sequence comprising yeast preferred codons. The IGF-IE sequence was inserted into pMON2690 in place of the IGF-Y sequence to create pMON2108 as shown in FIG. 2 Specifically, pMON2690 was digested with restriction enzymes BglII an dHindIII yielding two dsDNA fragments of pMON2690, one large and one small. These two dsDNA fragments were each isolated by electrophoresis in 0.8% (w/v) agarose gels (Maniatis et al., 1982) and thereafter eluted from the gels. The smaller dsDNA fragment containing the R.B.S., LAM and IGF-IY DNA sequence was then digested with restriction enzyme Ava2 and the about 210 base pair Ava2/HindIII fragment containing the IGF-IY sequence was then removed. The remaining Ava2/BglII fragment containing the R.B.S. and LAM sequences was then mixed, in the presence of T4 DNA ligase, with the large dsDNA fragment of pMON2690 generated and isolated as described above and a synthetic IGF-IE sequence containing an Ava2 sticky end at its 5'-end. The resultant plasmid was called pMON2108.
b. ompF
An expression plasmid containing the previously described ompF signal sequence in place of the lamB signal sequence in plasmid pMON2108 was constructed in a manner analogous to the method for pMON2108 construction described above (See FIGS. 3A and 3B). The resultant plasmid which contained, sequentially, a recA promoter, a G10L sequence, an ompF signal sequence, an IGF-IE coding sequence and a bacteriophage T7 gene 10 transcription termination sequence, was called pMON2123 and is shown in FIG. 3B.
Otherwise identical plasmids containing signal sequences derived from secreted non-outer membrane proteins in place of the lamB or ompF signal sequences were also constructed in accordance with the recombinant DNA techniques described herein.
E. coli strains SE6004, SE2060, W3110, JM101 and/or 294 were individually transformed (Maniatis et al., 1982) with one or each of the following expression vectors: pMON2690, pMON2108, pMON2123 or one of the otherwise identical plasmids containing a nonouter membrane signal sequence operably joined to an IGF-I DNA coding sequence. The IGF-I produced was then assayed for total IGF-I protein produced, accuracy of processing and biological activity in accordance with the previously described procedures. E. coli strain SE6004 was determined to be the preferred host for high level periplasmic production of accurately processed mature IGF-I. The lamB and ompF signal sequences were found to provide high levels (4-8% of total host protein produced) of accurately processed bioactive mature IGF-I in the periplasmic space. The non-outer membrane signal sequences failed to provide high levels of accurately processed mature IGF-I.
The foregoing examples illustrate preferred embodiments of the present invention and are not intended to limit the invention's scope in any way. While this invention has been described in relation to its preferred embodiments, various modifications thereof will be apparent to one skilled in the art from reading this application.
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|Fee payment
Year of fee payment: 4
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Owner name: MONSANTO TECHNOLOGY LLC, MISSOURI
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Auto enthusiasts know that luxury is built into each Volvo, and know that only the best replacement 850 parts will suffice when maintenance needs to be done. It's not a regular occurrence that you find yourself in need of a replacement PCV Valve to repair your automobile. Make no mistake, you bought your 850 because you understood you would be getting long-lasting quality and dependability both impeccably combined into one car, truck, or SUV. The vehicle in your driveway is head and shoulders above the rest when it comes to all the vehicles on the road today.
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SCOTT TWP. — The township's lone park could soon host weddings, family reunions and other celebrations.
Supervisors voted 3-0 at Thursday's meeting to hire A.J. Guzzi General Contractors to build a 40-foot by 80-foot barn-style pavilion at Scott Twp. Municipal Park for $42,598. In a separate motion, officials awarded P & R Construction Inc. the bid to build a concrete pad at the pavilion site off Route 632 for $13,200.
"We want to have a place that families can come and rent out for picnics, reunions or weddings," Chairman Michael Giannetta said. "Right now, we don't have anything like that at the park."
Construction on the pavilion is pegged for the fall, officials said. Once complete, it will feature picnic tables, restrooms and a kitchen, Supervisor Ed Hlavaty said.
He said a $23,000 grant via Lackawanna County's community reinvest program and a $5,000 donation from the Scott Twp. Lions Club will help cover the costs.
Also at Thursday's meeting, supervisors approved an agreement with the Lackawanna County Conservation District for a $36,762 maintenance project at Matechak Road. Mr. Hlavaty said he expects to start work on the road within the next two to three weeks.
"A lot depends on the weather, but we want to address it as quickly as possible," Mr. Hlavaty said.
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Oscar lifts Brazil to U-20 World Cup
BOGOTA, Colombia (AP) Oscar scored a hat trick to lead Brazil to a 3-2 win over Portugal in extra time on Saturday to win the Under-20 World Cup.
Oscar got the winner in the 111th minute, lobbing a shot from the right that was tipped by Portugal keeper Mika onto the underside of the crossbar before going in. He also scored in the 5th minute, and again in the 78th.
Alex leveled in the 9th minute for Portugal and Nelson Oliveira's 59th minute goal put the Europeans ahead. Brazil fought back to take its fifth U-20 title, having been the runner-up two years ago. Portugal was seeking its third title.
Brazil was the clear favorite for the match between former colony and colonizer, but it was an even contest from start to finish, with only the brilliance of Oscar making the difference.
Oscar broke through in spectacular style, sending a 40-meter (yard) free kick into the far corner, catching goalkeeper Mika flat-footed. It was the first goal yielded by Portugal in the tournament after six clean sheets in the previous matches.
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Oliveira set up Portugal's equalizer when he ran to the byline and cut the ball back across the face of the goal with Alex tapping it home.
Just a minute later, Brazil appeared to score, with a bouncing ball going over the Mika's head. Replays suggested the ball had crossed the line, but American referee Mark Geiger let play go on.
Brazil always looked more threatening in the first half but could not find a way through Portugal's formidable defense, crowding the front of the goal. The South Americans led in every statistical category in the first half and had 55 percent of the possession.
Oliveira put Portugal ahead in the 59th. He broke down the right, beat defender Juan and his shot from a sharp angle beat Brazil goalkeeper Gabriel at the near post.
Brazil's equalizer came in the 78th. Mika blocked the initial shot by Dudu, but Oscar slotted the ball home.
Brazil had a slight edge in possession over the 90 minutes and a 9-4 edge in shots on target, though Portugal often looked the more threatening.
Prior to Oscar's winner, the best chance in extra time fell to Portugal's Cateano, who broke down the right and forced Gabriel into a point-blank save, knocking the ball away with both hands.
The result avenged the result of the 1991 final, when Portugal defeated Brazil on penalties.
In Saturday's third-place playoff, Mexico defeated France 3-1.
Ulises Davila, Jorge Enriquez and Edson Rivera scored unanswered goals after Alexandre Lacazette had put France ahead with an 8th-minute header.
The third-place finish comes after Mexico's senior team defeated the United States in the final of the regional Gold Cup tournament in June, and Mexico won the Under-17 World Cup title in July.
Posted | Updated


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Oman Newborn, Baby & Maternity Photographer. Portrait & Lifestyle.
Category Archives: Blog
There is no excerpt because this is a protected post.
She sipped on iced tea while I scrolled through my newborn portfolio, the anticipation an excitement in her eyes. I knew right then that we had a connection, months before this sweetheart was born. As I do with all my newborn bookings, I booked her due date in my calendar and waited for "THE" message! […]
He was awake and alert and continued to feed. Then all of sudden he fell asleep and we got these amazing photos of sweet baby Tuna. Spiked up some hairs too! A cool boy in the making.
Two Little sisters born on the same month. A double shoot; Newborn with a Two year old Studio add on shoot.
Lovable, adorable, gorgous little man he was. A peaceful and smooth newborn studio shoot. Meet 12 day old Baby Khalid.


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Eliyahu Berligne
Eliyahu Berligne (Hebrew: אליהו ברלין, born 1866 died 25 February 1959) was a founder of Tel Aviv, an important member of the Yishuv in Mandate Palestine and a signatory of the Israeli declaration of independence.
Born in what is today Belarus in 1866, Berligne was an activist in Hovevei Zion and attended the First Zionist Congress. An opponent of the British Uganda Program, he visited Ottoman Palestine in 1905, and immigrated two years later, settling in Jaffa. After arriving, he founded an olive oil and soap factory in the Haifa and Gush Dan area, with the principle of Hebrew Labour.
A founder of the Progressive Party, Berligne later joined the General Zionists and was a member of the Jewish National Council. He served as treasurer of the council between 1920 and 1948,[1] as well as manager of Bank Hapoalim.
In 1948 he was amongst the 37 people to sign Israel's declaration of independence, although he was unable to attend the declaration ceremony due to illness.[2]
- ^ The Signatories of the Declaration of the Establishment of the State of Israel Israel Ministry of Foreign Affairs
- ^ Independence Testimonies Bring Israel's Birth to Life Haaretz, 27 December 2014
External links[edit]
- The personal papers of Eliyahu Berligne are kept at the Central Zionist Archives in Jerusalem. The notation of the record group is A308.
|This Israeli biographical article is a stub. You can help Wikipedia by expanding it.
- Jewish National Council members
- Members of the Assembly of Representatives (Mandatory Palestine)
- Signatories of the Israeli Declaration of Independence
- Zionists
- Hovevei Zion
- Progressive Party (Israel) politicians
- General Zionists politicians
- People from Mogilev
- Imperial Russian Jews
- Imperial Russian emigrants to the Ottoman Empire
- Israeli Jews
- 1866 births
- 1959 deaths
- Jews in Mandatory Palestine
- Jews in Ottoman Palestine
- Israeli people stubs


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BEIRUT/AMMAN (Reuters) – Kurdish militia pressed a big offensive against Islamic State in northeast Syria on Wednesday, cutting one of its supply lines from Iraq, as fears mounted for dozens of Christians abducted by the hardline group that recently beheaded 21 Egyptian Copts.
The Syriac National Council of Syria says Islamic State seized 150 Assyrian Christians from villages in Hasaka province in a mass abduction coinciding with the offensive in the same region by Kurdish forces backed by US-led air strikes.
Hundreds more Christians have fled to the two main cities in Hasaka province, according to the Syriac council and the Syrian Observatory for Human Rights, which is tracking the conflict.
Islamic State has killed members of religious minorities and Sunni Muslims who do not swear allegiance to its self-declared "caliphate". The group last week released a video showing its members beheading 21 Egyptian Coptic Christians in Libya.
The abductions in Syria follow advances by Kurdish forces against Islamic State in areas of the northeast near the Iraqi border – an area of vital importance to the group as one of the bridges between land it controls in Iraq and Syria.
"They want to show themselves strong, playing on the religion string, at a time when they are being hit hard," said Rami Abdulrahman, who runs the British-based Observatory, speaking by telephone.
The Syrian Kurdish YPG militia, backed by US-led air strikes, last month drove Islamic State from the Syrian town of Kobani, since when further signs of strain have been seen in the group's ranks.
The Assyrian Christians were taken from villages near the town of Tel Tamr, some 20 km to the northwest of the city of Hasaka. There has been no word on their fate. There have been conflicting reports on where the Christians had been taken.
"These were peaceful villages that had nothing to do with the battles," said Nasir Haj Mahmoud, a Kurdish official in the YPG militia in northeastern Syria, speaking by telephone from the city of Qamishli.


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Rock Candy Cakes
... Aug 2012
I'm curious on how MiVidaSweets uses Craigslist too.
view all »
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Lem's Driving School
5890 Bethelview Road Suite #9 Cumming, GA 30040
Certified Programs
Defensive Driving
Helpful Links
Contact Us
Welcome to Lem's Driving School
Lem's Driving School is a family owned business that has been serving the Metro Atlanta area since 2010. Our school serves the community with DUI and Risk Reduction classes and Defensive Driving/Driver Improvement courses. We hope that this website and our driving school will be able to help you accomplish your goals and serve you with the quality customer service you expect.
DUI/Risk Reduction Classes
Our DUI/Risk Reduction Program is certified by the Georgia Department of Driver Services (DUI-10181).
If you have been charged with any of the following:
DUI (Driving Under the Influence)
BUI (Boating Under the Influence)
Drug Possession
Other Drug Offenses
Underage Possession of Drugs or Alcohol
Georgia State Law requires attendance of a DUI/Risk Reduction program certified by the Georgia Department of Driver Services
Defensive Driving Classes
Our Defensive Driving Program is certified by the Georgia Department of Driver Services (DI-2180).
This program is perfect for
ticket dismissals
points and fine reduction
license suspension reinstatement
and insurance discounts
We provide Defensive Driving classes during the day and also at night.
Copyright 2013 Lem's Driving School LLC


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|<< Previous book
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Winter Wonders(2003)
(The sixth book in the Tales from Grace Chapel Inn series)
A novel by Melody Carlson
Louise's daughter Cynthia brings home a famed home and garden expert to write a children's book the Howard sister' peaceful lives are turned upside down. Alice realizes that the dogs at the racing track in a neighboring town are being mistreated, she gets involved in a cause much deeper than she intended.
Genre: Romance
Genre: Romance
Similar Books by other authors...
Used availability for Melody Carlson's Winter Wonders
See all available used copies of this book at Abebooks UK or Abebooks US
Hardback Editions
June 2008 : USA Hardback
|Title: Winter Wonders (The Tales from Grace Chapel Inn Series #4)
Author(s): Melody Carlson
ISBN: 1-60285-209-X / 978-1-60285-209-9 (USA edition)
Publisher: Center Point Publishers
Availability: Amazon Amazon UK Amazon CA
Genre Pages


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The Backpacks for Kids Foundation is a 501(c)(3) non-profit organization dedicated to providing children in need in the Greater Boston area with backpacks and school supplies for the upcoming school year. Backpacks for Kids was founded in 2001 by Kaitlin Murphy, Kristen Raymond, Kim Roth, and Leslie Wardwell. The organization was taken over by Elizabeth Allen, Eric Bartholomae, Sarah Bartholomae, and Devon Laidman, and it is now run by Charlie and Meredith Allen, Bailey Laidman and Kerrianne Brown, Co-Directors. Noel Gridley and Aryana Moghaddam are currently volunteering to prepare for their future role of Co-Directors, beginning in 2017.
Right Now
After the extreme success of this 2016 summer, providing 707 backpacks to children in need, we are now seeking donations and planning for the 2017 drive! We have maintained relationships with 10 different service agencies, and they have continued to rely on our support since 2001. Check out the blog for more updates!
Backpacks for Kids focuses on the one thing that children need in order to be successful: their education. You can help make a difference in a child's life.


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Hammock for sale
Expand Messages
- I have a Hennessy Ultralight Backpacker hammock for sale. (This is
the non-asymmetric hammock). I've used it maybe half a dozen times on
overnighters and it's in execellent condition - clean, no rips, no
wear showing. Weights are: Hammock and fly - 23.2oz. Stuff sack -
1.8oz. Tree Savers - 1.6OZ. Snakeskins - 3.5oz. The reason I'm
it is because I can't sleep well in it - I'm claustraphobic and also
not sleep in my back. Only $85 plus shipping (with snakeskins).
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I have 6 cavities on my upper teeth. My orthodontist wants to remove all of my braces and end treatment to fill the cavities
Is there any other way to keep them from removing my braces, i have spent so much money and time i cant stand to throw it all away like this. What should i do? Please help me.
Doctor Answers 1
These answers are for educational purposes and should not be relied upon as a substitute for medical advice you may receive from your physician. If you have a medical emergency, please call 911. These answers do not constitute or initiate a patient/doctor relationship.


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Spacious Flat by Melrose Avenue in West Hollywood!
Número máximo de hóspedes8
Tamanho1000 pé quadrado
Andar no.
- Recebe famílias (com crianças)
- Cozinha
- Lavadora / Secadora
- Estacionamento disponível
- Apropriado para Eventos
- Quarto com chave
- Animais de estimação permitidos
Resumo da propriedade
|North Curson Avenue, CA 90036 Los Angeles, CA, Estados Unidos
|Tipo de propriedade
|ID da Propriedade
|Taxa de aceitação
|Tempo de resposta
|Algumas horas
|Calendário atualizado
|2 semanas atrás
This cheerful and bright apartment is a welcoming resting spot for visitors to the city, and can sleep up to 8 guests comfortably. The kitchen is fully-equipped, while the original 1930s style bathroom has a separate shower and bathtub. A laundry room is located in the building.
Walk for 10 minutes' to The Grove, an upscale open-air center shopping and entertainment complex. Situated along 15 blocks of Hollywood Boulevard, the Hollywood Walk of Fame is 15 minutes' away by bus.
Visit the Hollywood Sign, an American cultural icon overlooking the city.
Regras da casaA $500 security deposit is required.
|Entrada após
|Check-in antes
|Por acordo
|Saída antes
|Política de Cancelamento
Comentários sobre a acomodação
Comentários sobre outras ofertas desse anfitrião.
Big and clean apartment (1st floor of a house), located in a pleasant residential area, in a quite central location to visit LA. The neighborhood is also convenient with the farmers' market, the Grove and many stores.
Perfect for 4 people, even 6 would be possible.
The House is located in a quiet and safe neighborhood and it is strategically situated next to Melrose Avenue with walking distanse to Hollywood Walk of Fame and Beverly Hills. But you need a car in L.A to get around anyway. The house is one of two apartments in a large house and you can hear your neigbours very well, especially if they are a having a party. The house is quite old and needs renovation, f.ex. is the heating system in the living room not reparable - no problem during most of the year, but quite cold during winter time, so the owner had to bring extra owens. The house has limited power capacity, so you have to be careful when you put all electricity on. The house is large an can easily host a family with 6+ and the owner brings extra beds as needed. For shorter stays and also during summer period, the house is probably great to rent.
It was a great experience and a great stay at your place.
I will come again.
It was impossible to contact the owner; she never answered our emails or phone calls. Finally got an email (After wimdu got involved) telling us that the key was in secure box by the door with a code for the box: Hellllo!!! the box was behind a locked screen door , impossible to get it. It took 1 1/2 hrs to sort this out on arrival after a long day in the air. A maintenance man finally open the door for us; we found him in a garage in the backyard. The place was spacious but property was questionnable; there was dust everywhere and it was obvious that the cleaning was done at a minimum.
The host cancelled the booking, this review is automated.
We were given a different house as the photos show, I believe the owner has more than one property in this area.
The house is spacious, the furnitures are old but workable. We arrived after 5pm but the cleaner has just began to clean the house, which wasn't very enjoyable as we had to wait for more than 1 hour to actually check in.
Overall it's alright although there are still lots of things can be simply improved. Good for group of 4 or more to stay.
Angie's place was great !!
Angie's place was great !!
Very nice house :)
- Qualidade do alojamento garantida
- Todos os anfitriões são verificados por telefone
- Pagamento seguro, certificado SSL


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Star Necklace
This cute little star necklace is available in silver, gold and rose gold. Mix and match the chain and star finishes for your own unique style. Star measures approximately 5/16 inch in diameter and hangs on a 17 inch chain.
Letter charm materials: 14k gold plated, 14k rose gold plated, and silver plated.
17 inch chain materials: 14k gold filled, 14k rose gold filled and sterling silver.
Your purchase comes in a beautiful glossy silver foiled box that is ready to give as a gift or to keep for yourself.
Unless otherwise specified, necklace chains are made 17 inches and bracelets are 7 inches. If you would like a longer length, please let me know in the "Gift Note" section at checkout. Lengths over 19 inches are $1.00 extra per inch. Special lengths are considered custom and are not refundable or exchangeable, so please be certain of the length and metal finishes you choose.
Please be sure to read our shop policies before purchasing.


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Click here to view sale items.
Can't find the item you are looking for using the search and browse tools above? View our special order catalog.
Profile Design's Airstrykes are so quick your competition won't know what hit them! Grab these light aluminum bars, get in a comfy, aero tuck and you'll fly faster with the same effort. Plus, the spring-loaded F-19 arm rests rotate up so you can quickly and easily access a comfortable climbing position. With these versatile bars your PRs don't stand a chance! Plus, the Airstrykes fit most handlebars thanks to Profile's ingenious Multi-Fit System.
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13 RV Parks near Telluride, Colorado
Mountain Village, Colorado 5.72 miles SE
Ophir, Colorado 10.36 miles SE
Ouray, Colorado 14.18 miles NE
Ridgway, Colorado 16.2 miles NE
2250 US-50 Telluride, Colorado 81416 (map) |
Sites: 41 | Latest Rate: $8.00 | 2 Reviews
|Clean Restrooms:
|Clean Showers:
Aug 2016 - $8.00
For those who want to "rough it" a bit. No electricity, water only from a spigot. Not a real lake; it's an irrigation reservoir but still kinda nice. The largest negative value of this campground (and the reason I didn't stay very long) are the hordes of evil, little spawn-of-Satan biting flies. They are way, way worse than mosquitoes and will keep you inside with window screens tightly closed most of the time. About half of the sites are level and large enough for a 30 footer; a few will acommodate big rigs. Nice views of nearby mountains. - Old Mountain Man
Oct 2012 - $14.00
Great remote location in the heart of the Rockies near Telluride and Wilson Mesa. This is a NSF site so not much in the way of amenities. I was there to shoot the fall colors but missed the window by a couple of weeks. Restrooms were closed, vault toilets, but they had portal potties at each of the restroom locations. Actually was able to connect via Verizon using a booster amp. But no over air TV. - Gary Camp-Inn
Priest Lake Road Ophir, Colorado 81426 (map) | (877) 444-6777
Sites: 28 | Latest Rate: $21.00 | 1 Reviews
|Clean Restrooms:
|Clean Showers:
Jul 2016 - $21.00
The most popular campground in the San Juan National Forest, and for good reason: the view is spectacular. Most of the sites are level or nearly so. All are gravel. Some sites are nicely shaded, others are in the sun.
Fills up quickly, even on weekdays. If you didn't make a reservation, you will need to get there in the morning to snag one of the first-come-first-served sites as someone else moves out. Cellular signal is too weak for handheld phones, but with a so-called "booster" antenna on an LTE router you can actually have Internet. - Old Mountain Man
US Hwy 550 - CR23 Ouray, Colorado 81427 (map) | (970) 325-4736
Sites: 120 | Latest Rate: $48.00 | 53 Reviews
|Clean Restrooms:
|Clean Showers:
Jul 2016 - $48.00
The good is the people were friendly, the grounds were clean and the restrooms were very clean. Otherwise this campground was not spectacular. The sites were all gravel except for some patches of grass. All sites had a table and firepit. There are lots of trees, and scenery is beautiful. Internet was strong but so slow. There was no pool - unusual for a KOA. There was no cable TV - sad, but not unusual. They have breakfast everyday - worth avoiding. They have BBQ on weekends - just so-so. Pizza on Mon/Tue was decent. They have music on weekends - bring your cowboy hat and boots and expect to dance and have fun. I rate this place average. Many KOAs are better, but there is little competition any better in the area; and there are several, Their rates are pricey by about $15. I would try another campground if I visit again. - Scot671
Jun 2016 - $49.00
Campground was very well maintained. We loved the streams within the park. We really enjoyed the office staff and found them to be very polite and helpful with any questions about the area. - RMahaney
Jun 2016 - $44.00
CG 5 miles north of Ouray actually. Typical KOA with lots of amenities - meals for purchase, recreation areas, etc. Large bathrooms with plenty of showers and stalls. Very nice staff. Lots of trees. Wonderful to hear the creek/river running near our site, although our site was a little tight. We would stay again. - Jolly Out There
790 Oak St Ouray, Colorado 81427 (map) | (970) 325-4418
Sites: 68 | Latest Rate: $42.00 | 49 Reviews
|Clean Restrooms:
|Clean Showers:
Sep 2016 - $42.00
Nice park with lots of trees and mountains surrounding the area. All your views are 'up'! My site was long enough I kept the 12' trailer attached and still had room to get the jeep into our sirte. - FOSJ
Sep 2016 - $35.00
This is a funky campground with a strange layout. The main section has the usual line of RV spots but across the (public) street are a line of E&W and non-serviced back in sites. We were in one of them. Had a tough time finding one that was level, but worked it out with blocks. We actually liked the site because it was very wide and had trees for privacy. The "riverside" sites weren't very impressive to me since the river is more like a canal running along the park with condos right on the other side. I certainly didn't think it was worth paying extra for them. The restroom and laundry room were clean and modern and showers worked well. The washers/dryers were a bit expensive but ok otherwise. There's also a folding table and a couple chairs. The best part of this place, as all others have mentioned, is that it's just a couple blocks from the center of town so no need to move your rig or unhook your tow/towed to go out for dinner etc. Nice. Wifi was mostly useless but Verizon worked well. I'd stay again. - Pdxjoanne
Aug 2016 - $45.00
We stayed in 4J for two nights. best place we stayed but mainly because of the location. We were able to walk into town, which was rare for most campgrounds.
The sites are very close together, have to juggle to put the awning up and have it over your picnic table, trying not to be too close to the sewage outlet! However the facilities were good and clean, showers free. WiFi very patchy and sometimes nonexistent.
Saying all that we would come back to the site, as it gave us everything we wanted. - Singaporeally
1700 North Main St Ouray, Colorado 81427 (map) | (970) 325-4523
Sites: 65 | Latest Rate: $48.00 | 23 Reviews
|Clean Restrooms:
|Clean Showers:
Aug 2016 - $48.00
This is a great park if you want to be very close to Ouray. The best thing going for the park is its location, just a mile from the main part of town. It's a quick drive to get there, or you can walk along the riverwalk trail which runs right along the RV Park. The park is also right along the Uncompahgre River. The views from the park are wonderful, though the tall mountains to the east mean that you won't see the sun until two hours later than official sunrise! The free WiFi worked quite well, Verizon worked well, but AT&T data was very slow. The park is on the expensive side (we paid $54/night after tax), and is mostly just a gravel lot with hookups with very little in the way of landscaping. It's also fairly noisy since it's right on Highway 550. We might stay again, but would try to find something a little less expensive nearby first. - LifeRebooted
Jul 2016 - $40.00
Overall very good, very crowded. But summer in Ouray is expected. Has large, wide, long spaces. Was gravel only, but easy access. - papa2
Jun 2016 - $55.00
One of our favorite parks ever. Friendly and super helpful owners and staff, and the greatest location. We chose a premium site which is right by the Uncompahgre River, and you can hear the rushing waters this time of year the moment you open your door. Great wifi, clean facilities, and within walking distance to town. We walked along the river several times a day as we made our way to the cute little town of Ouray. No bugs, AMAZING scenery, full hookups, close to town (about a 12 minute walk). Sites are gravel and fairly level. I can't say enough good things about this place, and we look forward to returning! - MikulasGoneAgain!
Highway 550 Ouray, Colorado 81427 (map) | (970) 874-6600
Sites: 31 | Latest Rate: $11.00 | 1 Reviews
|Clean Restrooms:
|Clean Showers:
Sep 2015 - $11.00
There are some sites that are suitable for small trailers -- not big rigs. Campground is very well located, directly above (literally) Ouray. Great views. Shady and cool. If you are not sure you can fit in here, better to scout the sites without your trailer first. - Prof Dan
664 Greene St Silverton, Colorado 81433 (map) | (970) 387-5512
Sites: 38 | Latest Rate: $40.00 | 12 Reviews
|Clean Restrooms:
|Clean Showers:
Jul 2016 - $40.00
The owners of Red Mountain RV are the best! There are many choices for an RV park in this beautiful area and Red Mountain is our home in Silverton. We stay in this park every Fourth of July and the Lockey's go out of their way to make us feel like family. - OutbackUSA
Jul 2014 - $43.00
This is a pretty average campground, but probably one of the best in the Silverton area. The campsites, reasonably level on a gravel bed, are fairly narrow and arranged in a double row, back to back. The utilities all work with no problem. The campground is fairly close to the railroad track, --- only this case it is a bonus --- as one can get a good view of the narrow gauge railroad trains driven by a coal-fired engine carrying sight-seeing passengers to and from Silverton two or three times a day. There are no trains at night! I am a little chafed at the high daily rate, as we were part of a large 4-wheeling rally. When we asked for Good Sam discount, we were told no discounts were given to rally participants --- not what we usually encounter when attending rallies. Still we would stay here again as the RV park is convenient to Silverton, and in a pleasant setting; but we would probably be a little more aggressive in trying to get a reasonable daily rate. - hoppytoad
Jul 2013 - $36.00
We stayed here for the 2nd time in the last 2 years. The pad sites are a little tight but very long. We had very good 50 amp and great water. The wife loves watching the trains come and go, and they love motorcycles Jeeps and 4 wheelers here. Will be back next year for the RMAR rally. Oh and the owners found a spare set of my motorhome keys and sent them to us, great caring folks. - notsofastdave
1445 Mineral St Silverton, Colorado 81433 (map) | (970) 387-9888
Sites: 17 | Latest Rate: $50.00 | 5 Reviews
|Clean Restrooms:
|Clean Showers:
Jul 2016 - $50.00
This park is now owned by Silverton Lakes Campground so you have to register there. I called ahead to confirm my site so I could go to A & B, unhitch the toad, and drive it to Silverton Lakes Campground to register. There is no one manning the office at A & B but we didn't need help. The nine sites facing southeast (toward Kendall Mountain) are far superior to the eight facing southwest. In addition to the views, the campground was adequate except for the WiFi, which cannot be counted on (Verizon signal was good). Cement Creek (the site of the infamous EPA spill) flows along the edge of the campground, but didn't look any different than when we stayed there a year ago. After checking out all the RV parks in Silverton, this one was the least crowded, quietest, and very close to the restaurants. - Dr. John
Sep 2014 - $40.00
This park is within 2 blocks of the main street of Silverton. Easy walk to any of the stores and restaurants and the D&S depot. At 9000+ elevation, it gets cold at night in September but the days warmed up nicely. Like everything else in Silverton, this park is a little overpriced but is quiet except when the trains arrive and depart. We will stay here again. - Delbert
Sep 2013 - $32.00
Top reasons to stay at A&B: 1) location. Located in the center of town. Sure, Silverton is small, but just 1 - 2 blocks away are restaurants, Montagna's Distillery and shops. 2) friendly folks. I have stayed here before and always feel good with these folks. My stay was somewhat open-ended and I had only booked 3 days with them but ended up staying 8 days (and my rate was adjusted to the weekly rate) but no problems! The park is not fancy but the views are great! - Nrf
640 Mineral Street Silverton, Colorado 81433 (map) | (970) 387-0240
Sites: 39 | Latest Rate: $38.00 | 10 Reviews
|Clean Restrooms:
|Clean Showers:
Oct 2015 - $38.00
We were two spaces west of office. Owners next door. It was just a week before they close for the season so park was almost totally empty. They are very nice, friendly and helpful. My wife called from Ouray as both it was raining and the road was closed during the day, opening at 5 PM. We wanted to know if they thought travel over the 550 was OK and was there snow at the top. Owner said road construction tight, but passable with our 40 footer towing. We would arrive after close and they were going to be out for the evening. Owner gave wife the Wi-Fi code, which space to park. We thought that was above what other parks have done.
He did come over and shut off our water at 5 AM, again at 10 AM, which the second time I saw him, opened the window to talk. He gave me a bit of a reprimand for a dripping hose connection. Says water is expensive in Silverton, one cent per gallon (maybe it is). I cost them about $3 for the 12 hours of drip. I apologized and the rest of our stay was great. We did arrive late and in a pretty good rain storm.
Wow what views there. The Durango/Silverton steam train is right behind the park so you see and here it every day. Park is walking distance to train station, and downtown Silverton. They have no fire rings but it's OK if you have your own.
Lots and lots of grass, a bit frosty in the morning which we were told is standard all summer. We will stay there again, but for more than the three nights as this time. - Bobcat1
Oct 2015 - $24.00
We had grass on 3 sides of us which was great for the dogs. Wifi was excellent and possibly the best I have ever had. I was unable to get the cable TV to work but that was not an issue because I prefer to use our satellite. This was our first trip to Silverton and hope to come back again. If we do we will definitely stay at Silver Summit. Parking site was on level gravel, electric was good and water was fine. This is not a resort nor does it pretend to be. Able to walk to anything in town. Excellent value with Passport America. - cubadoug
Jul 2012 - $30.00
This is our second year of staying here. The owners are so nice and always take the time to make your stay a pleasant one. There are a few trees scattered about and grass growing in most of the RV spaces. This park also offers Cabin rentals, Laundromat and showers. We loved the idea that this park is located on the far east of town but is still within walking (1/2 mile) distance of the downtown area. We chose this park because of the direct access to all off road riding. We have a 40' fifth wheel toy-hauler and needed somewhere we could unload the quads and ride directly from the park. This is the only RV Park in Silverton that allows that. The trail riding in this area is amazing and the summer weather temps are perfect. I would avoid this area during the holidays because the trails can get very congested. We will definitely come back to this park again. - shelliedoug
2100 Kendall St Silverton, Colorado 81433 (map) | (970) 387-9888
Sites: 47 | Latest Rate: $37.00 | 9 Reviews
|Clean Restrooms:
|Clean Showers:
Jul 2015 - $37.00
We had the same problem as the other couple that stayed July 2015. Park was packed and very difficult to maneuver RV and truck in. Luckily, on the morning we were leaving, the nice gentlemen across from us moved their trucks so we could pull out and leave. We have already reserved a more accessible camp spot for next year where we will not have this problem.
Staff was VERY nice. Location was great. Just outside of the main street. Made for a nice walk in the mornings. The roads to and from town were OHV friendly, so we could drive our UTV to the car wash and to the grocery store. They rent fire pits to you for very inexpensive, which was nice. They have 3 or 4 cabins that people without RV's can stay in.
We will be back next year but super happy we already chose and reserved our space. - 2TEXOMA
Jul 2015 - $37.00
After checking in, we had to ask around to have both an ATV and car moved because it would not have been possible to maneuver in to our site. The whole park is a muddy mess when it rains, but then again so is most of Silverton. What seemed to be the owners and their friends were being rather loud outside the main building near our site until about 11 PM. Short-term sites are very close together with slideouts practically touching. Our site was sloped up at the truck end which made detaching and attaching our weight distribution bars almost impossible due to the angle. Wi-Fi crashed and disappeared during the one time I used it. I probably wouldn't stay here again. On the bright side, the staff was friendly and helpful. - Brad & Angela
Aug 2014 - $36.00
The good: The park is a short walk into town and conveniently located. Seems to be more popular with the ATVers and off-road motorcycle crowd, as you can ride right out of the park and up to the trails. Our site was fairly level, the 50 amp power worked well and water pressure was great. There was a small grass "pet area" directly across from us that was also very convenient. The laundry room was clean although we didn't need to use it for anything more than getting a Wi-Fi signal. Staff was very friendly and helpful. The Bad: The entire park is part gravel, patches of grass here and there and mud. When it rains it's a bit of a mess so you had better have a patio rug to put outside your door. Wi-Fi signal was unusable even though we were parked within about 60' of the office. Had to go sit in the laundry room to be able to do anything. Some sites have the power and sewer on the wrong side requiring you to either park opposite direction of your neighbor or run all your hookups under your rig. This is a no frills park and it worked for our needs but I do wish they would bring in a lot more gravel to help with the mud situation. I would likely try and get into the park on the other end of town or dry camp at one of the wonderful locations that are so available in the area on our next visit to Silverton. - NwJeeper