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text:
|Publication number
|Publication type
|Application number
|Publication date
|6 Apr 1999
|Filing date
|7 Jun 1995
|Priority date
|4 Jun 1986
|Fee status
|Also published as
|Publication number
|Thomas M. A. Wilson
|Original Assignee
|Diatech Limited
|Export Citation
|BiBTeX, EndNote, RefMan
|Patent Citations (6), Non-Patent Citations (9), Referenced by (15), Classifications (55), Legal Events (4)
|External Links: USPTO, USPTO Assignment, Espacenet
This is a continuation of application Ser. No. 08/046,358, filed on Apr. 12, 1993 now U.S. Pat. No. 5,489,527, which is a continuation of application Ser. No. 07/148,650, filed on Feb. 4, 1988, now abandoned, which was filed as International Application No. PCT/GB87/00390 on Jun. 4, 1987, all of which are incorporated herein by reference.
1. Field of the Invention
This invention relates to enhancers of the translation of mRNA.
2. Discussion of the Background
The mechanisms by which eukaryotes and prokaryotes initiate translation are known to have certain features in common and to differ in others. Eukaryotic messages are functionally monocistronic, translation initiates at the 5' end and is stimulated by the presence of a cap structure (m7 G5' ppp5' G . . .) at this end (Shatkin, Cell 9 645 (1976)). Prokaryotic messages can be polycistronic, can initiate at sites other than the 5' terminus, and the presence of a cap does not lead to translational stimulation. Both eukaryotes and prokaryotes begin translation at the codon AUG, although prokaryotes can also use GUG. Translation in both is stimulated by certain sequences near the start codon. For prokaryotes, it is the so-called Shine-Dalgarno sequence (a purine rich region 3-10 nucleotides upstream from the initiation codon). For eukaryotes, it is a purine at the -3 position and a G residue in the +4 position (where the A of the AUG start codon is designated +1), plus other sequence requirements involved in finer tuning. This is part of the "relaxed" version of the scanning model (Kozak, Nuc. Acids. Res. 13, 857 (1984)) whereby a 40S ribosomal sub-unit binds at the 5' end of the eukaryotic mRNA and proceeds to scan the sequence until the first AUG, which meets the requirements of the model, is encountered, at which point a 60S sub-unit joins the 40S sub-unit, eventually resulting in protein synthesis. Reference can be made in this connection to the following publications by Kozak: Cell 15, 1109 (1978), Nuc. Acid. Res. 9, 5233 (1981) and Cell 44, 283 (1986).
Beyond these sequence requirements in and about the initiation codon, there are no additional regions of mRNA which are known to enhance translation reproducibly.
During eukaryotic transcription, sequences known as "enhancer" regions can provide transcriptional stimulation.
RNA viruses contain either positive or negative single-stranded RNA or double-stranded RNA. The class of RNA viruses include the majority of plant viruses (over 90%), some animal viruses and several bacteriophage.
One of the most widely studied of RNA viruses is tobacco mosaic virus (referred to hereinafter as TMV). The complete nucleotide sequence of TMV is known (Goelet et al., Proc. Natl. Acad. Sci. USA 79 5818 (1982)). The 5'-region of TMV RNA was first isolated in 1965 as an RNase T1 -resistant fragment 70 nucleotides long (Mundry, Z. Vererbungsl. 97, 281 (1965)) and free of internal G residues (Mandeles, J. Biol. Chem. 243, 3671). This region (later referred to as omega, Mandeles J. Mol. Biol. Chem. 243 3671) was sequenced (Richards et al. Eur. J. Biochem. 84 513 (1978)) and shown to form disome initiation complexes with wheat germ ribosomes. One ribosome occupies the AUG initiation side of the 126 KDa protein coding region and the second binds to the upstream leader sequence (Filipowicz and Haenni, Proc. Natl. Acad. Sci. USA 76, 3111 (1979); Konarska et al., Eur. J. Biochem. 114, 221 (1981)). Recent additional evidence to support the role of omega in ribosome association comes from the uncoating/gene expression data of Wilson and co-workers, (reviewed in J. Gen. Virol. 66, 1201 (1985) and UCLA Symp. 54 159 (1987)). It was observed in various in vitro translation systems that TMV particles are uncoated by the binding of ribosomes to the 5' end and their translational movement toward the 3' terminus.
It has now been found that the 5'-regions of RNA viruses act as enhancers of translation of mRNA.
According to a first aspect the present invention provides mRNA including the 5'-leader sequence of the RNA virus up to the first initiation codon of the viral RNA (or a derivative or portion thereof), the mRNA also including downstream sequence not derived from the virus providing the leader sequence.
According to a second aspect the invention provides a DNA sequence complementary to the 5'-leader sequence of an RNA virus, up to the first initiation codon of the viral RNA (or a derivative or portion thereof).
According to a third aspect the invention provides a DNA sequence comprising a promoter, a sequence complementary to the 5'-leader sequence of the RNA virus up to the first initiation codon of the viral RNA (or a derivative or portion thereof) and an open reading frame including an appropriate initiation codon. The invention also provides an expression vector including such a DNA sequence.
According to a fourth aspect the invention provides the use of the 5'-leader sequence of the RNA virus up to the first initiation codon of the viral RNA (or a derivative or portion thereof), either as RNA or as complementary DNA, as an enhancer of translation of mRNA when the 5'-leader RNA is contiguous with the mRNA.
FIGS. 1(A-B) Production of chimeric mRNA containing Ω';
FIG. 2 SDS-PAGE analysis of translation productions from MDL and WG;
FIG. 3 SDS-PAGE analysis of translation productions from E. coli;
FIG. 4 Chimeric RNA constructs containing mRNA and derivatives and portions of the Ω' leader sequence;
FIGS. 5(A-B) Chimeric RNA construct containing CAT mRNA with the 5' leader sequence from TMV (SPS isolate);
FIGS. 6(A-B) Chimeric RNA construct containing CAT mRNA with the 5' leader sequence from TYMV;
FIGS. 7(A-B) Chimeric RNA construct containing CAT mRNA with the 5' leader sequence from BMV RNA 3;
FIGS. 8(A-B) Chimeric RNA construct containing CAT mRNA with the 5' leader sequence from RSV;
FIGS. 9(A-B) Chimeric RNA construct containing CAT mRNA with the 5' leader sequence from A1MV RNA 4;
FIGS. 10(A-B) Chimeric RNA construct containing GUS mRNA (bad context) with the 5' leader sequence from pSP64 polylinker;
FIGS. 11(A-B) Chimeric RNA construct containing GUS mRNA (bad context) with the 5' leader sequence from U1-Ω;
FIGS. 12(A-B) Chimeric RNA construct containing GUS mRNA (good context) with the 5' leader sequence from polylinker;
FIGS. 13(A-B) Chimeric RNA construct containing GUS mRNA (good context) with the 5' leader sequence from U1-Ω;
FIG. 14 Effect of Ω' on the expression of capped or uncapped CAT RNAs in electroporated tobacco protoplasts;
FIG. 15 Effect of Ω' on the expression of capped or uncapped CAT RNAs microinjected into X. laevis oocytes;
FIGS. 16(A-D) Plasmids of pJII6 and pJII7;
FIGS. 17(A-E) Chimeric RNA constructs with the NPT II coding region and either Ω', two tandem Ω' regions, a long (77 base) "junk" leader (polylinker) sequence or "junk" together with a central Ω';
FIG. 18 Trp promoter modified to create a 3' Hind III site and a blunt 5' end;
FIG. 19 Constructs pUC35SCATNos and pUC35SVΩ'CATNos; and
FIG. 20 Graph showing expression of CAT in constructs pUC35SCATNos and pUS35SΩ'CATNos.
Most RNA viruses which have been sequenced contain 5'-leader sequences, i.e., sequences at the 5'-end up to but not including the first initiation codon. Appropriate 5'-leader sequences from any RNA virus may be used as a translational enhancer according to the invention.
As noted above, the 5'-leader sequence of TMV plus the AUG initiation codon is known as omega and its nucleotide sequence is known (Richards et al. Eur. J. Biochem. 84, 513 (1978)) so that cDNA can be prepared synthetically. The omega prime (Ω', see FIGS. 1A and 1B) sequence of TMV represents the preferred translational enhancer according to the invention.
Information is also available about the translational enhancement by 5'-leader sequences of other RNA viruses including the following:
Turnip yellow mosaic virus genome RNA (Filipowicz and Haenni. Proc. Natl. Acad. Sci. USA 76 3111 (1979)), brome mosaic virus RNA 3, alfalfa mosaic virus RNA 4 (Goelet et al., Proc. Natl. Acad. Sci. USA 79 5818 (1982)); Rous Sarcoma virus (J. L. Darlix et al. Nuc. Acids Res. 10, 5183 (1982)).
In any particular case it will almost certainly be possible to vary the natural 5'-leader sequence in such a way that its function as a translational enhancer is modulated but not destroyed. Such variants are referred to herein as "derivatives" and derivatives of the natural sequence and their use as translational enhancers form part of the present invention. It may also be the case that parts only of the natural 5'-leader sequence (or a derivative sequence) are required to provide translational enhancement. Such parts of the sequence are referred to herein as "portions" and portions of the natural sequence or of derivatives thereof and their use as translational enhancers form part of the present invention.
In any particular case, determination of the nucleotide sequence of the 5'-leader of an RNA virus should be within the expertise of a man of ordinary skill in the art using known and readily available techniques. Similarly, identification of those parts of the natural 5'-leader sequence which are essential for translational enhancement and those which can be modified or omitted (i.e. the identification of derivatives and portions of the natural sequence) is a matter which can be determined by routine experimentation.
The 5'-leader sequences of RNA viruses (or derivatives or portions thereof) can be used as such as translational enhancers according to the invention by ligating them upstream of an appropriate mRNA using for example T4 RNA ligase. The 5'-leader sequence may be immediately adjacent to the initiation codon of the downstream mRNA, or it may be spaced therefrom by an intervening sequence. However, it is preferred to use the 5'-leader sequence in the form of complementary DNA. The cDNA can be obtained in any manner which is desired. However in view of the relatively short length of the 5'-leader sequences in question it will usually be most convenient to synthesise the cDNA chemically. The cDNA will generally be incorporated into an expression vector. An expression vector is any vector capable of expressing those DNA sequences contained therein which are operably linked to other sequences capable of effecting their expression. An expression vector will usually contain a DNA sequence comprising a promoter, the sequence complementary to the 5'-leader sequence of an RNA virus up to but not including the first initiation codon of the viral RNA (or a derivative or portion thereof) and an open reading frame including an appropriate initiation codon. The open reading frame will generally code for one or more proteins of interest. The expression vector may be introduced directly into the host cell stably or transiently where the DNA sequence is expressed by transcription and translation. In the case of stable expression the vector must be replicable in the host either as an episome or as an integral part of the chromosomal DNA. Alternatively the expression vector can be transcribed in vitro and the RNA introduced directly into the cell for transient expression.
The translational enhancers according to the invention can be used in any situation where it is desired to enhance the translational efficiency of mRNA. However it should be noted that the effect of the enhancers is most marked in the case of an mRNA which is otherwise poorly translated. Thus the addition of either a cap or an enhancer according to the invention or both will stimulate translation. In the case of an mRNA already bearing a cap the effect of the enhancer according to the invention, is still present.
The translational enhancers according to the invention are effective in many different types of expression system, for example plant cells, bacteria and animal cells although a particular enhancer may be more suitable for one system rather than another. For example during testing in vitro the omega sequence of TMV appears to show a greater degree of enhancement in the wheat germ system and E. coli than in the rabbit reticulocyte lysate system although enhancement of translation is observed in all three systems. The matching of particular enhancers to particular expression systems will generally be a matter well within the expertise of the man of ordinary skill.
In general the translational enhancers according to the invention can be used with advantage in any case where the value of an organism or cell line to commerce, including agriculture, is determined by the level of expression of one of its natural genes or of an artificially introduced gene.
(i) In the manufacture of commercially useful proteins in genetically engineered organisms or cell lines.
(ii) In fermentation where the rate of production of a product of intermediary metabolism catalysed by enzymes can be enhanced by increasing the rate of synthesis of a particular enzyme.
(iii) In viral cross-protection where the effects of a viral infection can be reduced by prior exposure to an attenuated form of the virus and it is advantageous to increase the level of protection by increasing the level of production of the component which provides the protection. For example plants can be protected against infection by TMV by exposing them to a genetically engineered strain of Agrobacterium which contains a cDNA copy of that portion of the TMV RNA which codes for the viral coat protein. Bacterial cDNA inserted in the plant genome continually expresses the coat protein which protects the plant against infection by the virus itself.
(iv) In the genetic engineering of plants where a property such as herbicide resistance is installed by artificial modification of the intermediary metabolism of the plant.
(v) In the expression of a partial clone of a gene, for example where it is desired to raise antibodies to the product of expression of the partial clone.
The invention is illustrated further by the following experimental work.
Chimaeric mRNA according to an embodiment of the invention was produced in the following manner, as illustrated in FIG. 1.
Using the sequence of TMV RNA vulgare strain (Goelet et al., Proc. Natl. Acad. Sci. USA 79 5818 (1982)), an 81 base oligonucleotide was synthesised containing the DNA sequence complementary to the 67 base sequence upstream of the AUG initiation codon of the 126 kDa open reading frame, but not including the 5' cap (m7 Gppp and adjacent G residue). This sequence is called omega prime. A HindIII site was incorporated at the 5' end and a Sal I site at the 3' end of this oligonucleotide. A second oligonucleotide of 20 bases, including 13 bases at the 3' end of the omega prime sequence and a Sal I site, was synthesized as a primer for the synthesis of the second strand. After annealing, second strand synthesis was completed using the Klenow fragment of DNA polymerase I.
The resulting double-stranded 81 bp fragment was digested with HindIII/Sal I to create a 77 bp fragment with single-stranded cohesive ends and inserted into the corresponding sites of pUC19. The face that the synthesis had resulted in the correct orientation of the omega prime sequence of TMV was verified by sequence analysis using the dideoxy chain termination method (Sanger et al., Proc. Natl. Acad. Sci. USA 74, 5463 (1977)). The fragment containing omega prime of TMV was then introduced into the HindIII/Sal I sites of plasmid pJII1 to result in pJII101. pJII1 is derived by inserting the TMV origin of assembly sequence as a BamHI fragment into the corresponding site of pSP64 (Melton, Nuc. Acids Res. 12 7035 (1984)) and modifying the SmaI site to a Bgl II site (Sleat et al. Virology 155, 299 (1986)).
A 779 bp Sal I ended fragment from pCM1 (Close and Rodriquez, Gene 20, 305 (1982)) containing the chloramphenicol acetyltransferase gene (CAT) of Tn9 was introduced into the Sal I site of pJII1 and pJII101, resulting in pJII2 and pJII102, respectively. A 1.2 kb Bgl II/Sal I fragment from pKNR-H (Gallie et al., J. Bacteriol. 161, 1034 (1985)) containing the neomycin phosphotransferase II gene (NPTII) of Tn5 was made blunt ended with Klenow fragment and introduced into the Acc I site of pJII1 and pJII101 to result in pJII3 and pJII103, respectively. Correct orientation of both the CAT and NPTII genes was verified by restriction fragment analysis.
After linearisation of pJII2 and pJII102 with Bgl II and pJII3 and pJII103 with EcoRI, RNA transcripts of each were made using SP6 RNA polymerase. Capped versions of each were also made using GpppG and SP6 polymerase reaction conditions which ensure comparable yields of capped transcripts. Equivalent yields of RNA from the capped and uncapped reactions and for the constructs with or without the omega sequence were verified by denaturing formaldehyde-agarose gels.
Equivalent amounts of the various CAT containing transcripts and the NPTII containing transcripts were added to the rabbit reticulocyte lysate (MDL), wheat germ S-30 (WG) and E. coli S-30 in vitro translation systems under standard reaction conditions. The resulting polypeptide products were labelled with 35 S-methionine.
FIGS. 2 and 3 show the SDS-PAGE analysis of the translation products from MDL (FIG. 2, tracks 1 to 11). WG (FIG. 2, tracks 12 to 22) and E. coli (FIG. 3). In FIG. 2 template additions were: tracks 1 and 12, H2 O (endogenous translation); tracks 2 and 13, BglII-linearized pJII102 DNA; tracks 3 and 14, CAT RNA; tracks 4 and 15, omega prime-CAT RNA; tracks 5 and 16, 5'-capped CAT RNA; tracks 6 and 17, 5'-capped-omega prime-CAT RNA; tracks 7 and 18. EcoRI-linearized pJII103 DNA; tracks 8 and 19, NPTII RNA; tracks 9 and 20, omega prime-NPTII RNA; tracks 10 and 21, 5'-capped NPTII RNA; tracks 11 and 22, 5'-capped-omega prime-NPTII RNA. 14 C-labelled marker proteins (Amersham International, plc) were loaded on track M. Their respective sizes in kilodaltons (kDa) are shown on the left. The dried gel was autoradiographed for 3 days at room temperature. In FIG. 3 template additions were: track 1, H2 O (endogenous translation); track 2, BglII-linearized pJII102 DNA; track 3, CAT RNA; track 4, omega prime-CAT RNA; track 5, 5'-capped cat RNA; track 6, 5'-capped-omega prime-CAT RNA; track 7, EcoRI-linearized pJII103 DNA; track 8, NPTII RNA; track 9, omega prime-NPTII RNA; track 10, 5'-capped NPTII RNA; track 11, 5'-capped-omega prime-NPTII RNA. 14 C-labelled marker proteins were loaded on track M and their sizes (in kDa) are shown on the left. The dried gel was autoradiographed for 3 days at room temperature. It should be noted that although the E. coli S-30 system is a transcriptionally active system, the native E. coli RNA polymerase does not recognise the SP6 promoter.
The following Table 1 shows the stimulating effect of the omega prime sequence. Zones from gels shown in FIGS. 2 and 3 were excised and the radioactivity in each band of product of translation was measured and the data normalised in each case to that from RNA not containing the omega prime sequence. Expressing the data in this way eliminates any stimulating effect of the cap alone and shows only the stimulating effect of the omega prime sequence.
TABLE 1______________________________________mRNA WG MDL E. coli______________________________________CAT 1 1 1omega prime-CAT 10 1.2 11GpppG-CAT 1 1 1GpppG-omega prime-CAT 3.8 2.2 2.7NPTII 1 1 1omega prime-NPTII 71 17.8 74GpppG-NPTII 1 1 1GpppG-omega prime-NPTII 9.5 1.9 9.6______________________________________
The presence of the omega prime region of TMV stimulates the expression of transcripts containing either the CAT or NPT II sequences in either the capped or uncapped state. Stimulation was most marked with the translationally inefficient NPTII transcripts. Thus, the Tn5 fragment containing the NPTII gene contains an additional AUG immediately upstream from the AUG start codon of the NPTII gene. In addition, the "Kozak rules" (see the papers by Kozak referred to above) would suggest that the region about the NPTII start codon constitutes a poor signal for translational initiation. As soon in WG and MDL (FIG. 2, lanes 8 and 19), the NPTII transcript without either a cap or omega prime functions poorly. The addition of a cap or omega prime improves translation although the presence of omega prime has a greater stimulatory effect than the presence of a cap alone. E. coli translational machinery, although not cap-dependent, is greatly stimulated by the presence of the omega prime sequence (FIG. 3, lanes 4, 6, 9 and 11). In E. coli, addition of a cap to the omega prime sequence on CAT or NPTII mRNA did little to enhance its translation whereas addition of omega prime to capped CAT or NPTII mRNA resulted in greater enhancement.
CAT gene mRNA, even without a cap or the omega prime sequence, functions more efficiently in the eukaryotic systems. The first AUG from the 5' terminus is the start codon for CAT and the sequence in and about the start codon conforms well to the "Kozak rules". This is most evident in the MDL system where a much lower mRNA concentration was required to prevent exhaustion of the available 35 S-methionine pool. Not surprisingly then, the presence of the omega prime sequence in CAT mRNA was less stimulatory. MDL is thought less cap-dependent than WG, however the presence of a cap on the NPTII transcript had a slight stimulatory effect (FIG. 2, lanes 8 and 10), whereas its presence on the CAT transcript had little effect (FIG. 2, lanes 3 and 5). CAT mRNA alone was much less active in the WG system. As a result, the presence of either a cap or the omega prime sequence or both was highly stimulatory (FIG. 2, lanes 14 to 17). In WG, addition of a cap to the omega prime sequence on NPTII mRNA (FIG. 2, lanes 20 and 22) did little to enhance its translation whereas addition of omega prime to capped or uncapped NPTII mRNA (FIG. 2, lanes 19 and 21) resulted in a greater enhancement.
Therefore the presence of the omega prime sequence enhances translation in both eukaryotic (MDL and WG) and prokaryotic systems. This is consistent with the ability of TMV particles to be uncoated and the RNA translated by eukaryotic or prokaryotic translational machinery. The omega prime sequence often has a greater stimulatory effect on the expression of transcripts than does the presence of a cap alone. In addition, the omega prime sequence enhances translation under conditions where capping does not, e.g. in the prokaryotic and, to some extent, the MDL systems.
Further Experimental Work
Chimaeric RNA construct including CAT mRNA and derivatives or portions of the omega prime leader sequence were made in an analogous manner to that described above, the initial synthetic DNA sequence being chosen to produce the required RNA. As shown in FIG. 4, there were five deletions (referred to as Δ1 to Δ5), one A→C transversion and one C,A!n → U!n substitution.
Further chimaeric RNA constructs containing CAT mRNA were made in an analogous manner with 5'-leader sequences of viruses other than TMV (U1, vulgare, or common strain), namely the tomato strain of TMV (SPS isolate), Turnip Yellow Mosaic Virus (TYMV), Brome Mosaic Virus RNA 3 (BMV 3), Rous Sarcoma Virus partial leader (RSV) and Alfalfa Mosaic Virus RNA 4 (A1MV4). The initial DNA sequences synthesised and restriction site diagrams are shown respectively in FIGS. 5A to 9B.
A further set of RNA constructs was prepared in an analogous manner, but with the E. coli reporter gene β-glucuronidase (GUS) in place of CAT. The leader sequences used were either polylinker (for comparison purposes) or U1 omega (i.e. omega prime from TMV) or the derivatives and portions of omega prime and other viral leaders as described above. The GUS gene was used either in an artificial "good" Kozak context (see p. 33. re. Table 9) or in the native "bad" Kozak context as a SalI fragment from p RAJ235/240 (Jefferson et al. Proc. Natl. Acad. Sci. 83 8447 (1986)). The leader sequences and restriction site diagrams are shown in FIGS. 10 to 13. The expression of the reporter gene was determined fluorimetrically as described by Jefferson et al. Proc. Natl. Acad. Sci. 83 8447 (1986).
In addition to the three in vitro translation systems described above, two in vivo systems were used: microinjection of Xenopus laevis oocytes and electroporation of tobacco (Nicotiana tabacum cv Xanthi) mesophyll protoplasts. The respective techniques were as follows:
Two ng of each synthetic SP6 mRNA (or their corresponding DNA templates) were microinjected into the cytoplasm of stage 6 X. laevis oocytes in batches of 30 using standard procedures (Colman A. In Hames, B. D. and Higgins, S. J. (eds.). Transcription and Translation: A Practical Approach, IRL Press, Oxford 271 (1984)). Oocytes were incubated for 18 hours in Modified Barths' Saline (MBS), then washed briefly in distilled water. Extracts from Xenopus oocytes were prepared by resuspending each sample in 0.25 M Tris-HCl, pH 7.4, containing 10 mM DTT (17 μl/oocyte), followed by sonication for 15 sec. Insoluble material was removed by microcentrifugation for 15 min. and fractions of the resultant extract representing equivalent numbers of oocytes were assayed for CAT or GUS activity by published procedures (CAT: Gallie et al. Nuc. Acids. Res. 15 3257 (1987); GUS: Jefferson et al. Proc. Natl. Acad. Sci 83 8447 (1986)).
Mesophyll protoplasts were isolated from leaves of N. tabacum (cv. Xanthi) and stored in 0.7 M mannitol. The protoplasts (106) were centrifuged at 60� g for 3 min. resuspended in 1 ml ice-cold 0.7 M mannitol containing the RNA (8 μg), transferred to the electroporation cell and given a single voltage pulse (2.5 kV/cm) by discharging a 50 nF capacitor through the cell. The pulse had a rapid rise-time (less than 1 μs) and an exponential decay with a half-life of about 5 μs. Electroporated protoplasts were stored at 0-4� C. for 10 min. Ten mls of 0.7 M mannitol were added and the protoplasts recovered by centrifugation at 60� g. for 3 min. The protoplasts were then resuspended in culture medium and incubated at 25� C. for 21 hours in 9 cm-diameter Petri dishes.
Each sample of electroporated protoplasts was sedimented, resuspended and sonicated in 150 μl of 0.25 M tris-HCl, pH 7.4 containing 2 mM leupeptin and 10 mM dithiothreitol (DTT) for CAT assays or 50 mM sodium phosphate pH 7.0, 10 mM 2-mercapto ethanol for GUS assays. Extracts were microcentrifuged at 10,000� g, for 10 min at room temperature or 4� C.
FIG. 14 shows the effect of omega prime on the expression of capped or uncapped CAT RNAs in electroporated tobacco protoplasts. Each origin spot received extract equivalent to 5�104 viable protoplasts. For each sample, the conversion (%) of substrate (14 C-chloramphenicol) into its mono-acetylated forms (indicated on the left) is shown at the top of the track. Electroporated RNAs or standards were: track 1, no RNA (mock); track 2, CAT RNA; track 3, omega prime-CAT RNA; track 4, 5'-capped CAT RNA; track 5, 5'-capped-omega prime-CAT RNA; track 6, 0.1 unit purified CAT enzyme added to an equivalent volume of extract as for track 1; track 7, 0.1 unit purified CAT enzyme alone. The dried tlc plate was autoradiographed for 2 days before removing and determining the radioactivity in the relevant 14 C-labelled spots of mono-acetylated products (3-Ac/1-Ac), % conversion data reflects the relevant CAT activity in the extract. Omega prime (+/- a 5'-cap) was the most stimulatory of CAT RNA expression.
FIG. 15 shows the effect of omega prime on the expression of capped or uncapped CAT RNAs microinjected into X. laevis oocytes. An equal amount of each CAT RNA-construct or linearized DNA template (2 ng) was injected per oocyte, throughout. Equal oocyte extract volume (equivalent to 0.3�cell) were assayed in each case, unless stated otherwise. Conversion (%) of 14 C-chloramphenicol into its monoacetylated forms, is shown at the top of each track. In tracks 5 and 6, conversion (%) into the di-acetylated form is indicated by *. Microinjected RNAs or standards were: track 1, no RNA (mock); track 2, BglII-linearized pJII102; track 3, CAT RNA; track 4, omega prime-CAT RNA; track 5, 5'-capped CAT RNA; track 6, 5'-capped-omega prime-CAT RNA; track 7, as for track 5 but 20-fold diluted extract; track 8, as for track 6 but 20-fold diluted; track 9, 0.1 unit purified CAT enzyme added to an equivalent volume of extract as in track 1; track 10, 0.1 unit purified CAT enzyme alone. The dried tlc plate was autoradiographed at room temperature for 18 hours, before excising and counting the relevant 14 C-labelled spots.
The following Tables show the further results obtained.
TABLE 2______________________________________Effect of deletions and other sequence alterations ontranslational enhancement by omega prime. Chloramphenicolacetyltransferase expression in RNA-microinjected Xenopusoocytes. % conversion of .sup.14 C- chloroamphenicol to the 1- andRNA injected 3- monoacetylated forms______________________________________Nil (mock) 0.1CAT 4.5Omega prime CAT 34.2Δ1 - CAT 17.2Δ2 - CAT 9.7Δ3 - CAT 17.6Δ4 - CAT 14.6Δ5 - CAT 17.9A -> C transversion - CAT 15.7 C, A!.sub.n -> U!.sub.n substitution - CAT 34.30.1 units CAT (pure enzyme) 1.2______________________________________ All RNA's were uncapped
The above results show that omega prime enhances translation of CAT. Deletions 1 to 5 reduce this effect without destroying it entirely. The same is true of the A→C transversion. The U!n substitution has little effect, if any.
TABLE 3______________________________________Effect of untranslated 5'-leader sequences fromdifferent viruses on the level of expression ofchloramphenicol acetyltransferase after microinjection ofmRNA into Xenopus ooctyes. % conversion of .sup.14 C-chloroamphenicol to the 1- and 3 - monoacetylatedRNA injected forms______________________________________Nil (mock) 0.1CAT 6.3Omega U1 CAT 46.5Omega (SPS) CAT 35.8TYMV CAT 1.3BMV3 CAT 5.0RSV CAT 5.4AlMV4 6.70.1 Units CAT (pure enzyme) 1.3______________________________________ All RNAs were uncapped Omega U1 derives from the standard strain of TMV (Vulgare). TYMV: turnip yellow mosaic virus leader. BMV3: brome mosaic virus RNA3. RSV: a partial leader sequence from Rous Sarcoma virus. AlMV4: alfalfa mosaic virus RNA4.
The above results show that omega prime from TMV U1 enhances translation of CAT RNA in this system. TMV tomato strain omega prime also does so, but a little less well. Other viral leaders have little or no effect, TYMV leader may be inhibitory.
TABLE 4______________________________________Effects of different 5'-non-translated leader sequenceswith or without a 5'-cap on in vitro translation ofchloramphenicol acetyltransferase mRNA in a wheat germ cell-free system. (The results as shown below have beennormalized to the unmodified CAT mRNA leader sequence whichwas takem as 1.0 to show fold-stimulation over CAT mRNAalone).RNA added Uncapped capped______________________________________CAT 1.0 1.0Omega prime CAT 2.8 3.7Δ1 - CAT 1.2 0.6Δ2 - CAT 2.0 1.8Δ3 - CAT 1.5 1.3Δ4 - CAT 3.0 1.5Δ5 - CAT 1.5 0.7A -> C transversion-CAT 3.3 2.9 C, A!.sub.n -> U!.sub.n substitution-CAT 2.1 3.4TMV Tomato strain Omega prime-CAT 1.7 3.0TYMV-CAT 0.3 3.0BMV3-CAT 1.4 2.8RSV-CAT 0.6 2.5AlMV4-CAT 2.1 2.3______________________________________ Note: these figures represent comparisons of proportional stimulations of translation produced by the inclusion of different sequences at the 5' en of the CAT message. Translations were done with capped or with uncapped messages. Each column represents the yield of radioactive CAT protein recovered by excision from an independent gel (as in FIGS. 2 and 3). The bands were cut out, counted, and have been normalized to a band for which the mRNA was unmodified CAT message.
The above results show that omega prime enhances in this system, with or without 5'-capping. The other viral leader sequences and derivatives or portions of omega prime enhance translation to varying extents, but rarely significantly more than omega prime itself.
TABLE 5______________________________________Effects of different non-translated leader sequences,all without a 5'-cap structure, on in vitro translation ofchloramphenicol acetyltransferase mRNA in an E. coli cell-free system.RNA added Uncapped______________________________________CAT 1.0Omega prime CAT 9.0Δ1 - CAT 3.3Δ2 - CAT 2.5Δ3 - CAT 6.3Δ4 - CAT 4.2Δ5 - CAT 3.8A -> C transversion CAT 4.1 C, A!.sub.n -> U!.sub.n substitution CAT 1.9TMV Tomato strain Omega CAT 6.6TYMV CAT 0.2BMV3 CAT 3.7RSV CAT 1.5AlMV4 CAT 1.4______________________________________
The above results show that omega prime, some of its derivatives, and tomato strain omega prime enhance translation to the greatest extent. Most of the other sequences also enhance, but to varying extents. E. coli does not require a 5'-cap for translation.
TABLE 6______________________________________Effect of different leaders on the translation of β-glucoronidase mRNA (from pRAJ235/240) electroporated intotobacco protoplasts.GUS mRNA nmoles 4-methyl umbelliferyl Foldconstruct glucuronide cleaved min.sup.-1 /μg Stimula-used* protein in protoplast extract tion______________________________________ Blank! (no mRNA) -- --Uncapped Leader235 SP6 transcript, <0.01 1polylinker leader.!Omega prime (U1) - 235 0.25 >25Omega prime (SPS) - 235 0.15 >15TYMV - 235 <0.01 --AlMV4 - 235 <0.01 --BMV3 - 235 <0.01 --RSV - 235 <0.01 --Capped Leadercapped 235 SP6 0.03 1transcript polylinkerleader!capped Omega prime - 235 0.54 18capped Omega prime 0.43 14(SPS) - 235capped TYMV - 235 <0.01 --capped AlMV4 - 235 0.23 8capped BMV3 - 235 0.23 8capped RSV - 235 0.21 7capped Δ1 - 235 0.55 18capped Δ2 - 235 0.50 17capped Δ3 - 235 0.32 11capped Δ4 - 235 0.54 18capped Δ5 - 235 0.54 18capped A -> C transversion - 0.69 23235capped C, A!.sub.n -> U!.sub.n <0.01substitution 235 (blank value subtracted from all others)______________________________________ (*Constructs were "bad" GUS ex pRAJ 235/240, with or without a 5' cap)
All mRNA constructs were electroporated at 8 μg/ml and washed protoplasts incubated for 20 hours as described (Gallie et al. Nuc. Acids. Res. 15, 3257 (1987)).
The sequence of the "Bad" Kozak construct (235) leader is as follows: ##STR1## and the deleted omega prime derivatives, etc. follow accordingly.
Messenger RNAs encoding calf preprochymosin or chicken prelysozyme were synthesized in vitro, with or without the TMV 5'-terminal sequence (omega prime). The mRNAs were translated in vitro in the systems derived from rabbit reticulocytes (MDL), wheat-germ (WG) or E. coli (EC).
The plasmids pJII6 and pJII7 (FIGS. 16A-16D) were constructed using components of plasmids pSP64CT and pSP64LT (Sleat et al. Virology, 155 299 (1986)) and pJIII01. Both pJII6 and pJII7 were derivatives of the transcription plasmid pSP64 (Melton, 1984), which contains a promoter for the bacteriophage SP6 RNA polymerase adjacent to the polylinker sequence derived from M13 mp11. The preprochymosin and prelysozyme coding regions of pSP64CT and pSP64LT respectively were excised as HindIII fragments, blunt-ended with the Klenow fragment of DNA polymerase 1, and cloned into the HincII site of pJIII01 to generate pJII6 and pJII7 respectively (FIG. 16). These constructs were used to direct the synthesis of messenger-sense preprochymosin and prelysozyme transcripts bearing a 5'-terminal omega prime sequence. Parental plasmids pSP64CT and pSP64LT were templates for synthesis of mRNAs lacking omega prime.
The results as shown in Table 7 below.
TABLE 7______________________________________Effect of omega prime on expression of two eukaryoticmRNAs in 80S - and 70S - ribosome - based translation systems.Incorporation of L- .sup.35 S!-methionine (cpm) into gel-purified polypeptides.Cell-free system CPM (Rel. Stimulation)S26 transcript MDL WG E. coli______________________________________Prelysozyme 5040 (1.0) 3730 (1.0) 0 (1.0)omega prime prelysozyme 5290 (1.0) 10240 (2.8) 1030 ( )Preprochymosin 2470 (1.0) 1110 (1.0) 0 (1.0)omega prime preprochymosin 7280 (3.0) 10010 (9.1) 2180 ( )______________________________________
In an analogous manner to the methods described above, various chimaeric RNA constructs were prepared with the NPT II coding region, and either omega prime, two tandem omega prime regions, a long (77 base) "junk" leader (polylinker) sequence or "junk" together with a central omega prime. The constructions are shown in FIGS. 17A-17E and the results in Table 8 below:
TABLE 8__________________________________________________________________________EFFECT OF VARIOUS 5'-LEADER CONSTRUCTIONS ON IN VITRO TRANS-LATION OF SP6 TRANSCRIPTS OF NEOMYCIN PHOSPHOTRANSFERASECell-free Translation System Rabbit reticul- Wheat Germ ocyte lysate E. coli (-cap) (+cap) (-cap) (+cap) (-cap) (+cap)__________________________________________________________________________NPTII 1* 1 1 1 1 1omega prime NPTII 8.1+ 5.9 1.3 0.8 15.2 4.8 omega-omega!prime NPTII 8.2 6.3 1.1 0.7 44.9 15.0"JUNK"-NPTII 0.9 0.4 0.1 0.1 3.5 2.3"JUNK"-omega prime NPTII 1.8 1.1 0.3 0.3 9.8 8.2__________________________________________________________________________ *Background (H.sub.2 O or linearized DNA) corrected/All values = Fold stimulation over NPTII RNA alone.
This shows that a random polylinker sequence will not substitute for omega prime in 80S ribosome systems and only poorly in a prokaryotic (70S) system. Also two tandem omega primes only benefits 70S expression (c.f. single copy of omega prime).
To extend the data presented in Table 6 (above) on the effect of omega prime on expression of the β-glucuronidase mRNA (GUS mRNA), a new series of constructs utilized the GUS gene with a modified sequence context around the initiation codon (AUG). Plasmid pRAJ275, containing the "good context" leader ##STR2## was the source for this version of GUS. Omega prime and various other leader constructs were inserted at the Hind III/Sal I sites as before. Details of the pRAJ275 construct are as follows:
pRAJ275 is a derivative of pRAJ255 (Jefferson, et al. Proc. Natl. Acad. Sci. 83, 8447 (1986)) in which the 5' sequences of GUS were removed by progressive BAL31 deletion and replaced with a synthetic oligonucleotide that has constructed a "consensus" translational initiator as defined by Kozak (Microbiol. Reviews. 47, 1 (1983)). This plasmid has a unique Nco I site (CCATGG) positioned at the initiator ATG codon, with the surrounding context GTCGACCATGGTC. It has been shown both in vitro and in vivo that the context around the initiator can have a profound effect on the levels of translated product from a given amount of mRNA. This construction has been adjusted to maximise this effect. It was provided as a Sal I-Eco RI fragment in pUC19.
5'-capped or uncapped transcripts were electroporated into tobacco protoplasts as before and the level of GUS expression measured in extracts.
Uncapped RNAs were also added to MDL or WG cell-free translation systems and the level of GUS protein synthesized measured by excising and counting radioactive gel bands as for FIGS. 2, 3 etc. (Table 1). The results are shown in Table 9.
TABLE 9__________________________________________________________________________Translational Enhancement by omega prime on GUS mRNAIn vivo translationElectroporated tobacco protoplasts Gus activity nmoles 4-methyl umbelliferyl In vitro translation glucuronide MDL WG cleaved min.sup.-1 /μg Cpm .sup.35 S Cpm .sup.35 S protein in Fold methionine- Fold methionine- FoldKozak protoplast stim- labelled GUS stimu- labelled GUS stimu-context extract ulation n gel bands lation in gel bands lation__________________________________________________________________________UncappedRNAsGUS 235bad <0.01 (1) 7180 (1.0) 2400 (1.0)(40b poly-linkerleader)Omegabad 0.18 (>18) 19700 (2.7) 9720 (4.1)prime-235GUS 275good <0.01 (1) 12920 (1.0) 3010 (1.0)(28b poly-linkerleader)Omegagood 0.35 (>35) 29280 (2.3) 19720 (6.6)prime-2755'cappedleaderGUS 235bad 0.03 (1)(40b poly-linkerleader)Omegabad 0.61 (20)prime-235GUS 275good 0.04 (1)(28b poly-linkerleader)Omegagood 3.2 (80)prime-275__________________________________________________________________________
A further set of RNA constructs was prepared, in an analogous manner to the method described above, with the E. coli reporter gene β-glucuronidase (GUS) and additionally the inducible trytophan (TRP) promoter. The Trp promoter was purchased from Pharmacia Ltd., and modified by conventional techniques of restriction endonuclease digestion, filling-in, sub-cloning etc. to create a 3'-Hind III site (FIG. 18) and a blunt 5'-end. This dsDNA fragment was then inserted into the various leader-reporter (GUS) gene plasmids between the SP6 promoter (silent in E. coli in vivo) and the 5'-leader construct of interest.
The resulting plasmid DNA was used to transform competent E. coli cells by standard techniques and the Trp promoter induced in situ to produce chimaeric leader-reporter mRNAs. Transcription start point corresponds to the G in the Hind III site (AAGCTT).
Expression of the transcripts in transformed E. coli cells was determined spectrophotometrically (Jefferson et al. Proc. Natl. Acad. Sci. 83, 8447 (1986)). Results (Table 10) indicate that expression of GUS was enhanced in vivo from either the "good" or "bad" Kozak context construct by the presence of omega prime and to some extent by the other leader sequences or derivatives of omega prime.
TABLE 10______________________________________Expression controlled by the Trp promoter in stablytransformed E. coli cells Approximate relative spectro- photometric rates of conversion of p-nitrophenyl glucuronide______________________________________235-GUS ("Bad" context) 1omega prime 235 GUS 7x275-GUS ("Good" context) 1omega prime-275-GUS ("Good" 8xcontext)Δ1 -275-GUS 5-8xΔ2 275 GUS (75x: To be confirmed)Δ3 275 GUS (75x: To be confirmed)Δ4 275 GUS 5-8xΔ5 275 GUS 5-8xΔ -> C 275 GUS (75x: To be confirmed) C,A!.sub.n -> U!.sub.n 275 GUS 5xTYMV 275 GUS 3-4xAlMV4 275 GUS 3-4x3MV3 275 GUS 1-2xRSV 275 GUS 1-2x______________________________________
(The results above were normalized to the respective GUS construct with the polylinker leader alone.)
Omega prime enhances the expression of GUS RNA in vivo in E. coli transformed with plasmid DNAs, using Trp promoter in situ.
Further experiments were performed using supercoiled double standard pUC19 DNA which is modified to contain the cauliflower mosaic virus (CaMV) 35S promoter. The nopaline synthase terminator (from Agrobacterium tumefaciens) and in between them either CAT DAN (, ▴) or omega prime CAT DNA (∘, Δ).
The constructs (FIG. 20) were prepared from a binary vector system (Bevan, Nuc Acids Res. 12 8711 (1984)) wherein the CaMV 35S promoter and the Nos terminator were excised using Hind III and Eco RI sites and inserted into plasmid pUC19 thus giving pUC35S Nos.
The constructs pUC35SCATNos and pUC35S omega prime CATNos were made by inserting either CAT or omega prime CAT respectively into the plasmids using the Bam HI. site.
Various levels of either plasmid DNA (in μg/ml) were electroporated into protoplasts as before, and the cells incubated at 25� C. for 6 hours (∘, ) or 26 hours (Δ, ▴) and lysed. CAT assays (as before ) were carried out.
The results as seen in FIG. 20 indicate that cells which received 100 μg-150 μg of supercoiled plasmid DNA showed enhanced CAT expression (2 fold) when omega prime is present in the construct (Δ, ∘).
These results show that gene expression in stable transgenic plants will also likely be enhanced by the inclusion of the omega prime sequence between the promoter (for transcription in vivo) and the translational open reading frame.
|Cited Patent
|Filing date
|Publication date
|4 Dec 1985
|11 Apr 1989
|Massachusetts Institute Of Technology
|Process for enhancing translational efficiency of eukaryotic mRNA
|25 Aug 1994
|14 Nov 1995
|Mycogen Plant Science, Inc.
|Subgenomic promoter
|6 Nov 1984
|3 Jul 1985
|Teijin Limited
|Gene fragments derived from human immunoglobulin gene
|16 Aug 1985
|22 Apr 1992
|BEHRINGWERKE Aktiengesellschaft
|Enhancer for eukaryotic expression systems
|Title not available
|Title not available
|Brederode et al, "Complete Nucleotide Sequence of Alfalfa Mosaic Virus RNA 4", Nucleic Acids Research, vol. 8, No. 10, pp. 2213-2223 (1980).
|Brederode et al, Complete Nucleotide Sequence of Alfalfa Mosaic Virus RNA 4 , Nucleic Acids Research, vol. 8, No. 10, pp. 2213 2223 (1980).
|Gehrke, "Enhancing the Translation Efficiency of Plant Messenger RNAs", Abstract No. PO-1-147 in The Book of Abstracts from the First International Congress of Plant Molecular Biology, Savannah, 27th Oct.-1st Nov. 1985.
|Gehrke, Enhancing the Translation Efficiency of Plant Messenger RNAs , Abstract No. PO 1 147 in The Book of Abstracts from the First International Congress of Plant Molecular Biology, Savannah, 27th Oct. 1st Nov. 1985.
|Lewin, Genes II, 2nd Edition, 1985, John Wiley & Son, NY, p. 165.
|Pelletier et al, "Insertion Mutagenesis to Increase Secondary Structure Within the 5' Noncoding Region of a Eukaryotic mRNA Reduces Translational Efficiency," Cell, vol. 40,515-526, Mar. 1985.
|Pelletier et al, Insertion Mutagenesis to Increase Secondary Structure Within the 5 Noncoding Region of a Eukaryotic mRNA Reduces Translational Efficiency, Cell, vol. 40,515 526, Mar. 1985.
|Ravelonandro et al, "Structure of the 5'-Terminal Untranslated Region of the Genomic RNAs from Two Strains of Alfalfa Mosaic Virus", Nucleic Acids Research, vol. 11, No. 9, pp. 2815-2826 (1983).
|Ravelonandro et al, Structure of the 5 Terminal Untranslated Region of the Genomic RNAs from Two Strains of Alfalfa Mosaic Virus , Nucleic Acids Research, vol. 11, No. 9, pp. 2815 2826 (1983).
|Citing Patent
|Filing date
|Publication date
|21 Jul 1999
|16 Oct 2001
|Large Scale Biology Corporation
|Method for conferring herbicide, pest, or disease resistance in plant hosts
|21 Jul 1999
|30 Jul 2002
|Large Scale Biology Corporation
|Method of compiling a functional gene profile in a plant by transfecting a nucleic acid sequence of a donor plant into a different host plant in an anti-sense orientation
|6 Jun 2003
|19 Dec 2006
|Keio University
|Recombinant template used for producing a carboxy-terminal modified protien and a method of producing a carboxy-terminal modified protein
|23 Oct 2006
|30 Jun 2009
|Dow Agrosciences Llc
|Preparation of vaccine master cell lines using recombinant plant suspension cultures
|1 Jun 2009
|21 Jun 2011
|Dow Agrosciences Llc
|Preparation of vaccine master cell lines using recombinant plant suspension cultures
|27 Dec 2007
|21 Jul 2015
|Rodina Holdings S.A.
|Artificial DNA sequence with optimized leader function in 5′ (5′-UTR) for the improved expression of heterologous proteins in plants
|19 Aug 2009
|10 Nov 2015
|TET Systems GmbH & Co., KG
|Tetracycline inducible transcription control sequence
|26 Jan 2001
|4 Sep 2003
|Teemu Teeri
|Use of a nucleotide sequence for enhancing protein synthesis and expression of proteins
|16 Jul 2004
|6 Jan 2005
|Large Scale Biology Corporation
|Method of determining the function of nucleotide sequences and the proteins they encode by transfecting the same into a host
|6 Jun 2003
|13 Jan 2005
|Hiroshi Yanagawa
|C-terminal modified protein and method for producing the same, modifying agent and translation template used for producing C-terminal modified protein, and method for detecting protein interaction with use of C-terminal modified protein
|27 Dec 2007
|17 Feb 2011
|Universita 'degli Studi Di Udine
|Artificial dna sequence with optimized leader function in 5' (5'-utr) for the improved expression of heterologous proteins in plants
|19 Aug 2009
|6 Oct 2011
|TET Systems GmbH & Co., KG
|Tetracycline inducible transcription control sequence
|26 Jan 2001
|2 Aug 2001
|Licentia Ltd.
|Use of a nucleotide sequence for enhancing protein synthesis and expression of proteins
|26 Jan 2001
|10 Jan 2002
|Licentia Ltd
|Use of a nucleotide sequence for enhancing protein synthesis and expression of proteins
|27 Dec 2007
|10 Jul 2008
|Universita' Degli Studi Di Udine
|Artificial dna sequence with optimized leader function in 5' (5'-utr) for the improved expression of heterologous proteins in plants
|U.S. Classification
|435/69.1, 435/419, 435/325, 435/252.3, 536/24.1, 435/70.1
|International Classification
|Cooperative Classification
|European Classification
|21 Mar 2002
Owner name: COPERNICUS TECHNOLOGY LIMITED, VIRGIN ISLANDS, BRI
Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNOR:DIATECH LIMITED;REEL/FRAME:012707/0053
Effective date: 20020315
|29 Aug 2002
|Fee payment
Year of fee payment: 4
|8 Sep 2006
|Fee payment
Year of fee payment: 8
|9 Sep 2010
|Fee payment
Year of fee payment: 12

subset: commoncrawl


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Low-quality hay causes long-term problems
Even if the drought was ending shortly, the effects on your beef herd wouldn't end with it.
That's because after a drought, it can take up to 2 years for cows and their calves to recover from the effects of subsisting on drought-shortened hay, namely in how heifers develop in late summer before calving in the winter, says University of Missouri Extension beef nutritionist Justin Sexten. And, that's exactly what happened last year, causing affected heifers to lose body condition scores and resulting in taking away body conditioning in the back and hip area.
"Unfortunately, much forage harvested last season does not meet base requirements," Sexten says. "During July and August, when no grass grew, heifers mined condition off their back."
A heifer should be at 85% of her mature body weight or above in order for her adequately support her calf after birth. Sexten says if a cow's lighter than that and her body condition score is at 5 or below (out of 9), she'll likely have trouble supporting a calf on her own colostrum, something that can get the young animal started off weakly or, at its worse, lead to a short lifespan.
These issues are propagated in a year like 2012 by a forage supply that's low in volume and protein, Sexten says. For example, a gestating cow needs a steady supply of hay with at least 10% protein. And, with what hay that's out there now -- at least that grown in an area under drought this year -- a lot of it will fall short of the basic nutrient requirements for a heifer or bred cow. And if that's the case, you'll have to supplement it with additional nutrients.
"The only way to know hay's nutrient content is with a forage test," Sexten says. "Marginal hay requires supplementation."
So how will you supplement your hay? Take corn gluten, for example. An additional 4 pounds of gluten per head per feeding will carry additional cost now, but "you can pay now or you pay later," Sexten says.
"If a heifer loses her calf, a $2,000 replacement heifer becomes worth $900 in salvage value," according to a university report. "A heifer in poor body condition after calving likely will not rebreed to calve the second year. That happens often, causing huge losses for beef herd owners. A lost heifer must be replaced. "Loss of a calf or heifer puts hay and grain prices in new perspective."


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Tag: surfer bracelets
The Journey of Bekl LifeStory Colourful Charms
The Journey of LifeStory Colourful Charms…
We've had a lot of interest in the LifeStory charms from Bekl recently. Now that summer is coming, we thought we'd share with you the secret of our beads, and what makes them special charms. The truth is, these charms have been on a journey all of their own by the time they get to your wrists, and we thought this would make interesting reading for you all!
The colourful charms start their life in Greece. Artisans make each and every bead BY HAND using local clay. The clay is sculpted into 'round' discs and put in a kiln to bake. Once the beads are 'cooked' and cold, the charms are covered in a colourful glaze by hand. This means that each charm created for Bekl is totally unique. The beads often have their own little characteristics – often finger prints and chips too, adding to their character. Sometimes the glaze also 'chunks up' meaning the thickness of beads changes from charm to charm. It's actually very charming 😉
But that's only half the story… once this process has been completed, the charms are then shipped from our artisans in Greece, to good old Blighty, where they are worked on in our Derbyshire workshop! The beads are individually drilled with our diamond drill bit, then polished by hand too… Once all that's done, we lightly sand each of the colourful charms by hand to give that 'seaworn' look. And just like magic, your colourful charms are ready!
So as you can see, it's not just a case of 'we buy the beads and sell them on.' These colourful charms, from the moment they are 'born' are individually crafted by hand at every step of the process. By the time they reach your wrist, they've been on a little journey of their own.
Not only do our charms represent your lifestory through their colours, but they also have stories of adventure to tell of their own, and it's all written into their design…
Make sure you're following us on instagram, twitter and facebook to keep up to date with the latest news, launches and offers!
2 weeks until LifeStory Launch
The Lifestory Bracelets launch in 2 weeks time and we thought we'd share a sneak peek of the bracelets with you…
Above, are the natural leather examples of the bracelets. We have three colours available – natural with gold ends, brown with gold ends and black with black ends. A charm bracelet for both guys and girls!
The beauty of the bracelets is that each charm can be bought seperately and represents a different emotion, inspiration or motivation, meaning you can literally WEAR your daily motivation. You can also use the bracelet to coordinate your outfit!
The charm bracelets are made of the highest quality leather and use strong magnetic closures, making them the perfect gift and surf inspired charm bracelet.
The LifeStory collection of charms and bracelets will go on sale on 30th January 2015… We hope you enjoy the range, more images to follow!


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(The authors are Reuters Breakingviews columnists. The opinions expressed are their own.)
By Neil Unmack and Richard Beales
LONDON/NEW YORK, Dec 28 (Reuters Breakingviews) -
( Anticipated publication around 2020.)
The most surprising thing about the demise of two-and-20 hedge fund fees is how long it took. Neither losses in the 2008 credit crunch nor the feeble returns during the ensuing seven-year euro zone crisis did much to bring down the archetypal fee structure: 2 percent management fee and 20 percent of gains. Now, though, it can finally be said: two-and-20 is dead.
Back in September 2012, the average hedge fund still charged 1.6 percent annually in management fees and collected 18.7 percent of any gains, according to data provider Preqin. Through November that year, the average global hedge fund investor earned just 2.6 percent, according to the HFRX global index maintained by Hedge Fund Research. In 2011 investors lost nearly 9 percent. The average annual return from 2009 to 2012, supposedly recovery years following the losses of more than 20 percent in 2008, was a measly 3 percent.
A few investors – including huge institutions like California's giant pension fund, Calpers – managed to negotiate better deals. More often, funds that did badly simply shut down, while investors often signed up for new funds at the usual fees in the hope that the high-return strategy so elegantly set out for them by the latest persuasive trading titan turned out to work.
And a few duff years did little to dent demand. Hedge fund assets reached $2.2 trillion by the third quarter of 2012, some 17 percent above the 2007 pre-crisis peak. By 2015 they were still rising.
Yet it now looks as if those traumatic years marked the beginning of the end for the image of hedgies as superhuman traders. Faced with unpredictable markets, heavy central bank intervention and increasing red tape, some managers simply gave up. Pioneer Stanley Druckenmiller closed his fund in 2010, noting tellingly that managing more than $10 billion didn't allow him to make the kind of returns he wanted. Some others did the same or returned money to outside investors. A wave of insider-trading scandals further tarnished the sector's image, casting doubt along the way about the legitimacy of some top managers' returns.
It didn't help that hedge funds were becoming an industry. After the 2008 collapse, regulators and investors alike watched funds more closely. Managers needed to have large businesses to justify the required information and compliance systems, and diverse ones to avoid concentrated risks that might scare cautious institutional investors like pension funds. As Druckenmiller had foreseen, big firms ended up making lower returns, on average. And for the fund firm bosses, management fees approaching 2 percent made a large-scale business a ticket to riches even without stellar returns.
Investors initially settled for modest returns during good years and capital protection in bad times. But then many realized that the two-and-20 fee structure was out of kilter. Pension fund managers' own rising liabilities made them tougher in claiming a higher proportion of the investment returns on their money. The most sophisticated ones set up their own in-house managers, staffed with refugees from shrunken investment banks and unlucky hedge funds.
Now in 2020, there's still a lot of money run by hedge funds. But there's a clear bifurcation. It's hard to distinguish parts of the industry from traditional asset managers like BlackRock (BLK.N) and Fidelity – and those behemoths have snapped up some funds. In this branch, firms can still charge a management fee of 1 percent. But performance fees have shrunk to 5 percent or less, often after a minimum risk-free return is reached, in recognition of the more earthbound expectations investors now have of performance.
The other wing of the industry consists of hedge funds that have essentially gone back in time to something closer to the original concept – impressive returns in all market conditions, with fees to match as long as the returns are forthcoming. In the 1960s, Warren Buffett ran a fund charging no management fee but taking 25 percent of investment gains above a 6 percent threshold return.
In 2012 Guy Spier, whose $100 million-plus Aquamarine Capital Management has a class of shares that follows Buffett's old structure, referred to the "microscopically small proportion of investment managers who decline to charge a management fee". Eight years on, there are more of them, though it's the harder and lonelier – if arguably the most investor-friendly – of the two hedge fund roads. Neither would look familiar to a manager who retired hugely wealthy in the early part of the past decade. But as places to invest, at least both now have fees that are a better fit.
- For previous columns, Reuters customers can click on [UNMACK/] and [BEALES/]
(Editing by Richard Beales and Martin Langfield)
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1947 Decatur High School DECANOIS, Decatur, Illinois VG
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Sixty years ago this 1947 DECANOIS was published for the students of old DECATUR HIGH SCHOOL, Decatur, Illinois. The cover is ivory colored with red lettering on the front cover. The yearbook is in excellent condition considering age. The spine is strong. All pages are intact. Coach Gay Kinter was the basketball coach, baseall coach and athletic director. Geral Cox was the tennis coach. Dennis Berkey was head football coach and David Batthauer was his assistant. Francis A, ("Smock") White coached track. Among the football team were Jim Hazelrigg, Bob Kerwin, Dick Lockmiller, John Krigbaum, Ciney Rich. On the basketball team were Don Galka, Ciney Rich, and Roger Brady. Most popular seniors were Mary Maxwell and Richard Lockmiller. Best looking couple was Arlene Kiemer and Bill Rose. Most likely to succeed were Phyllis Bridgewater and James M. Butts. Ads showing the Varsity Theater, A.E. Staley Mfg. Company, Phillips Dairy and many more bring back memories...... Buyer prepays by check, MO or by credit card through PayPal and adds $5.00 postage, packaging and insurance if mailed to a U.S. address. Visit our new eBay store from Decatur and Springfield, llinois - LINCOLN LAND MERCANTILE.


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I think this is just a happy, bring a smile to your face card! Have a wonderful day! :)
A woman who works with her hands is a LABORER; a woman who works with her hands and head, a CRAFTSMAN; but a woman who works with her hands, her head and her heart is an ARTIST. ~ Louis Nizer
Tuesday, January 12, 2010
Button Up!
This was a fun and quick card to make, using my new button stamp from "Square Pegs" on a Kraft base and a hodgepodge of real ones from the neutrals and new Gina K Romance Assortment! I started by stamping a random border with the button image in Memento Tuxedo Black and Versamark (difficult to see in this pic but I embossed the lighter ones, great to have that realistic shine and texture!) and then adhered the real buttons to create a collage using pop dots and glue dots. Last I added sime faux stitched over the button holes with my white Inkssentials and black glaze pens, the greeting and "free" pierced some little curvy trails in between to add more whimsy. Edges of card are rubbed with Cream White to soften the look. TIP: Don't have the color buttons you want? Stamp the button image and punch out with different size circles (or even a small squre!) to create some new buttons!
Crafted with YOU in mind by Melanie at 1/12/2010 07:15:00 AM
Labels: Gina K Designs, Square Pegs
7 comments:
- Tina Gilliland said...
Cute cute!! Can't wait to get my set!
- January 12, 2010 at 7:58 AM
- Sandra said...
love it!
- January 12, 2010 at 9:29 AM
- Jennie said...
Love this card Mel! I knew the button would be so fun :o) Definitely on my list to get soon!
- January 12, 2010 at 10:59 AM
- Ravengirl said...
What a fun, fun card!
- January 12, 2010 at 11:48 AM
- Anita said...
Cute card! Great tip!
- January 12, 2010 at 2:05 PM
- Robin McK said...
This is completely adorable!
- January 12, 2010 at 2:26 PM
- Elaine M said...
You're right - this one does make ya smile....love it
- January 12, 2010 at 4:26 PM
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At Gemvara, we believe that the jewelry you wear should be as unique as you. Every beautiful Gemvara Palladium Round Black Diamond Antique-Inspired Jewelry design will be made just for you. Gemvara uses only the highest quality materials to craft your Palladium Round Black Diamond Antique-Inspired Jewelry with meticulous attention to detail. Our precious metals are recycled or responsibly mined. Black diamond has a unique dark glamour.A platinum group metal, palladium has high purity and white tone of platinum but is less dense, making it more affordable.Our wide selection of Palladium Round Black Diamond Antique-Inspired Jewelry by talented designers means you can find rings, earrings, and necklaces that are exactly your style. You can also shop by style, choosing our Bars, Hearts, or Modern Collections. When our work is done and you open that box, you'll see your design is even more beautiful when brought to life in the finest precious materials.
We can help right away. Chat with a jewelry expert now!


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Discrimination "arising from" disability
We provide a mid-year round-up of key employment law cases in 2017.
In this week's podcast, we predict the key cases for 2017. We explain why employment status in the gig economy will be a big talking point, and flag up a major equal pay case against a private-sector employer.
We discuss a number of recent cases that concern NHS employers but that could also apply more widely.
We explain how three common scenarios can lead to a tribunal claim for discrimination arising from disability.
On this week's XpertHR Weekly, we discuss a recent Employment Appeal Tribunal case dealing with "discrimination arising from disability".
Audio and video: HR and legal information and guidance relating to discrimination "arising from" disability.
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Reed Business Information Limited trading as XpertHR is an Appointed Representative of Abbey Protection Group Limited which is authorised and regulated by the Financial Conduct Authority.
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Tere Calloway has a diverse list of schools involved in his recruitment and this summer the Seattle (Wash.) Seattle Prep three-star cornerback could start taking visits.
The 5-foot-11, 185-pound prospect who will attend the B2G Elite Camp at Cal Lutheran this week already has offers from Colorado, Idaho, Memphis, Montana, Nevada, Portland State, Purdue, San Diego State, SMU, Southern Miss, Syracuse, Temple, Tulane and Wyoming.
Trips will be important as Calloway looks for the best option.
Article Continues Below
"I haven't been on any visits yet but this summer I'm hoping to visit a few schools but I'm not sure if it's going to happen," Calloway said.
"Right now I'm in the works with visiting Memphis and Vanderbilt. I'm also looking at Southern Miss and Tulane and then I'm also looking at Nevada and if North Carolina State offers I'll take a visit there."
Calloway, rated as the No. 63 cornerback in the 2013 class and seventh in the Washington state rankings by Rivals.com, said he's been receiving lots of interest from Boise State, NC State, Oregon State, Washington State, Arizona State and others.
Since so many more things can happen in his recruitment for the remainder of the summer and into his senior season, the Seattle Prep standout does not want to name any frontrunners just yet.
"I'm still talking to a whole bunch of schools," Calloway said.
"I wouldn't say there are any favorites yet. There are definitely some schools stepping it up with me. Recruiting-wise, there are some schools that are recruiting me the hardest but other than that I haven't named any favorites yet."
Calloway shined at a 7on7 tournament this spring in Las Vegas. A big performance at the B2G Elite Camp can only help his stock as the summer continues.


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Best of 2014 #8 TV/Film: Honey Maid Showers 'Love' on Haters of its Inclusive Ad
Negative Feedback Becomes Something Beautiful in New Film out of Droga5
Published on Apr 03, 2014
Editor's Pick
Through next week, we continue our countdown of the best work of the year in TV/Film, Print/Outdoor/Design and Interactive (IX)/Integrated/Social. See all the Best of 2014 so far here.
In March, graham cracker brand Honey Maid, along with agency Droga5 redefined what "wholesome" means in a spot that celebrated families of all kinds, including gay couples and single parents. As you can imagine, the ad earned both praise and criticism, the latter which the agency turned into the heartwarming message of strength and hope you see in the film here.
Original Post:
Last month, Mondelez graham cracker brand Honey Maid set out to redefine the idea of "wholesome" with a much-buzzed about ad that highlighted a diverse range of families.They included a gay couple and their son as well as a family with a white father, black mother and mixed-race kids. The ad, not suprisingly, drew out a lot of haters on social media. But Honey Maid, along with agency Droga5, has taken all that ill-will and turned it into something beautiful---a massive paper sculpture forming the word "Love." Even the positive feedback -- which was ten times that of the negative -- was incorporated into the piece as a large sea that encompasses all that transformed "hate."
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Dean Foods Closes Sale of Morningstar Foods Unit to Saputo
Dean Foods Company (NYSE: DF) today announced the completion of the previously announced sale of its Morningstar Foods division to Saputo Inc for US$1.45 billion.
As a result of this transaction, Dean Foods expects to realize approximately US$887 million in proceeds, net of taxes and expenses. The Company plans to use all net proceeds toward fully retiring its remaining senior secured term debt, significantly lowering its leverage and increasing its financial flexibility.
Dean Foods also completed the previously announced transaction with The WhiteWave Foods Company (NYSE: WWAV), a controlled subsidiary of the Company, whereby WhiteWave received $60 million net of taxes as consideration for the termination of an option to purchase plant capacity and property at a Morningstar facility and the sale to Morningstar of certain manufacturing equipment located at another Morningstar plant.
Posted-In: News Asset Sales
© 2017 Benzinga.com. Benzinga does not provide investment advice. All rights reserved.


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STILLWATER, Minn. (WCCO) — Twice a month, WCCO-TV gets to do something pretty fun — surprise someone who's helping others. This week's Making a Difference winners are Suzanne Rode and Lisa Rambo who started Every Monday Matters.
They were volunteering at Valley Outreach Food Shelf in Stillwater when WCCO's Amelia Santaniello surprised them with the award.
Once the shock wore off, Rode and Rambo explained how they turned a hard day of the week into a special one.
"Instead of having Mondays be the day I start my diet or the day that I dread of the week, I'm gonna make Every Monday Matter," said Rambo.
A year later, Rode and Rambo are still coming up with new ways to help friends and strangers alike.
"Some nights we make seven meals, some nights … we made up to 25 meals in one day … and then we go and just deliver them and surprise people," said Rambo.
Writing letters for troops — they've done it. Shoveling snow for strangers — done that, too.
Rode said they definitely get something out of it.
"It fills us up, tremendously to give back to other people," she said.
"It's raining today, I'm going to go to Wal-Mart and walk people to their car, so they don't get wet," said Rambo. "Sometimes it's a lot of work, and sometimes it's no work, it's just seeing a need and filling it."
What's really amazing is how these two manage to fit in service with everything else on their plate. They work. Rambo has four kids and Rode's got three.
Still they find time to help other people.
For Making a Difference, they received $500 from American Equity Mortgage and WCCO. The pair was nominated by Rambo's sister.
WCCO-TV's Amelia Santaniello Reports


text: The scratch is such a common problem, experienced by many (an oops moment) but mostly by people new to 1911s. Spend a little time on the 1911 Forum and learn how many people have experienced or want to avoid the problem. Newbies, like I was, learn the correct way to reassemble and pick up a few easy tips (credit card, tape, etc.) to avoid the scratch. The reassembly scratch is easier to accept than other scratches and dings because it doesn't indicate misuse. I just wouldn't want one on a high-end pistol unless I put it there.


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Top 10 reasons to do family research
Expand Messages
- A nice article to share with your relatives that can't quite set aside the
time to help you with family research.
Past-Present-Future: Are you on the Fence? Top 10 Reasons to Jump into
Family History!
Have you been tempted to start researching your family history but just have
not gotten motivated to do so? Does genealogy seem overwhelming? Are you
not sure where to start?
It's time to get off the fence. Don't wait! Don't leave for tomorrow what
you can do today! Here is my advice as to why:
Jim Benedict
(courtesy Marian Pierre-Louis, Google+ Stream of 10 Feb 2012)
__________ Information from ESET NOD32 Antivirus, version of virus signature
database 6878 (20120212) __________
The message was checked by ESET NOD32 Antivirus.
Your message has been successfully submitted and would be delivered to recipients shortly.


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Logan & Mason
New Season Designs Have Landed
This season, be inspired by bedlinen fresh off the Logan & Mason design floor. Featuring bold prints, vibrant patterns and tactile embellishments, this range of quilt covers and accessories is a fresh and inspiring nod to contemporary style. There are fun and whimsical motifs that nurture and embrace your child's creativity, glamorous and exotic designs with a boho-luxe vibe, fresh contemporary flora for a more feminine feel, plus classic stripes and aquatic shades to suit a coastal theme. Be sure to pair with the co-ordinating cushions and throws for an expertly styled look.
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November 19, 2017
The environmental research center for scientists, students, and families interested in the natural world.
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Carnegie Museum of Natural History is building a new website! Look for it in 2017.
Biodiversity Studies in Pennsylvania
Pennsylvania Barrens Habitat Invertebrate Study: Year 3
Funded by PA Wild Resource Conservation Fund. The invertebrates and associated flora of six different types of barrens communities have been documented in the field (1996-1997). During 1998 Museum experts will determine and database the extensive collections now being prepared in order to assess differences and similarities among these habitats. The study will improve understanding of these unusual communities and will address how to effectively manage and maintain the unique biodiversity they contain. Collaboration with Botany at Carnegie Museum of Natural History, Western Pennsylvania Conservancy, and The Nature Conservancy.
Pittsburgh BioBlitz '98A 24-hour biological survey of Pittsburgh's Frick Park to focus media and public attention on biodiversity, 15 May 1998. A total of 1,164 species were documented in Pittsburgh's Riverview Park during the Pittsburgh BioBlitz '97, including a rare species of longhorn beetle, Obrium rubidum (Coleoptera: Cerambycidae). Of all species found, 64% were invertebrates in the Riverview Survey, and 55% were insects (of which 30% were beetles). Because most of life on Earth is invertebrate, most invertebrates are arthropods, most arthropods are insects, and most insects are beetles! Carnegie Museum of Natural History entomologists will have their hands full, especially the coleopterists!
Our eNews features information on exhibitions, upcoming events, and subscriber-only special offers!
Carnegie Museum of Natural History
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If you own a Samsung Galaxy S II and are a fan of MIUI interface custom ROMs for Android a new alpha version of the smartphone has now landed. The ROM is only in alpha stage now and the devs are very clear that you can't expect a fully functional ROM right now. The devs do give a nice list of what is working and what isn't working and the list of what's not may keep all but the most ardent tinkerers away right now.
Right now the ROM will boot up, shows the screen, hardware keys, internal storage, and SD card as well as offering functioning Bluetooth. What isn't working and are still on the to-do list includes the boot logo, the ability to get a phone and data connection to make the phone actually work, and WiFi. Various sounds and other items are still missing as well.
If you want to put the alpha ROM onto your S II, you can download the file needed on the XDA-developers forums and there are complete install instructions too. The install instructions include reboot to CWM, wipe the cache, data, system, and Dalvik cache and install the ROM form a SD card. You need to select MIUI-test.zip as well. When a fully functional ROM will be offered is unknown. You can see the boot process of an S II running the alpha ROM on the video below.
[via XDA-Developers]


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John West 'marathon' by BJL
AdvertiserJohn West
ExposureUnited Kingdom and Europe
A marathon runner dressed in a latex tuna suit is the star of the John West TV ad for its Tuna Light Lunch snacks. The ad, which was filmed at the Prague Marathon, opens with epic shots of the event. However, the seriousness of the race is undermined when the favourite, a Kenyan runner, is beaten to the finishing line by the sudden appearance of the giant rubber fish. The TV ad, by BJL, kicks off a campaign aimed at persuading women of the nutritional value of a John West Light Lunch. Direct marketing and in-store activity are supporting the TV advertising.


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that she really enjoyed getting to know the people, the science, and the business of biotech, moreso than other projects she might have worked on. In 1992, Maderis chose to join one of the few hot biotech companies run by a businessman rather than a scientist—Henri Termeer. She joined Genzyme that year in corporate development, and within a year worked her way up to vice president and business manager of the company's gene therapy program for cystic fibrosis.
This was a fascinating project for anyone from Harvard or Bain. Cystic fibrosis is a genetic disease caused by a single malfunctioning gene, which causes a buildup of thick sticky mucus in patients' lungs, and usually kills people before the age of 40. Researchers thought that since the specific gene had been identified for cystic fibrosis, it stood to reason that this disease out to be cured by gene therapy. The concept sounded simple—attach a properly functioning copy of the gene to a virus which would efficiently shuttle it inside cells and do its job.
Longtime biotech followers know it wasn't that simple. There is no cure today for cystic fibrosis, and there is no gene therapy yet approved by the FDA for sale in the U.S.
But there were still fascinating things for Maderis to learn on this project. She had to work on corralling all sorts of intellectual property for the whole gene therapy package, which hadn't been done before. The business strategy was being crafted at a time when gene therapy was essentially unregulated. If Genzyme was going to be successful in gene therapy, then Maderis needed to learn a lot more than just the dynamics of the market, physicians, pricing models, and the competitive landscape. It forced her to engage with regulators to talk through the issues, to testify before Congress when lawmakers had their inevitable concerns.
"Henri was visionary in saying it's a new industry, the science is always going to be one step ahead of where the regulation is, and we need to help craft it, and help people understand it, so we can craft an appropriate business climate and regulatory environment to support what is a very dynamic and new industry," Maderis says.
As the years went by in the late '90s, the next big thing was genomics and the highly publicized Human Genome Project. Maderis conceived of a division at Genzyme, called Molecular Oncology, which had a tracking stock that went public in 1998 on the idea of using genomic data for cancer. Maderis worked out two dozen partnerships with many of the big names in Big Pharma, oversaw 100 employees, and told the company's story to Wall Street.
By 2003, Genzyme had given up on the idea of tracking stocks, and consolidated everything under its corporate banner. This was also year after Enron, Worldcom, and other corporate scandals gave rise to the Sarbanes-Oxley Act, generally making it clear to Maderis that life at a public company "had a lot of downsides."
That was the year she was wooed back to California. Lewis T. "Rusty" Williams, the former head of R&D at Chiron, along with investors at Kleiner Perkins Caufield & Byers, Versant Ventures, and Texas Pacific Group recruited her. The cast of wooers included none other than VC legend Brook Byers himself. The concept was to build a new protein drug discovery engine, based on $70 million of prior R&D from the Japanese government, for a company called Five Prime Therapeutics. Williams and Maderis knew each other "by reputation," Maderis says, as leaders at fellow big biotech companies. Maderis also wanted to return to the Bay Area to be closer to her mother, who was in failing health. So she said yes to her first experience as a biotech startup CEO, and joined in September 2003, the same time the company raised $12 million in its second round financing.
"It was about the ability to get in on the ground floor in a company with exciting technology, build out the platform, a pipeline, and provide huge value to Big Pharma in partnerships. It was an interesting model," Maderis says.
Maderis stayed at Five Prime until … Next Page »
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New Exclusive Lead System for the Moving and Relocation Industry
Rank My Web LLC offers a custom business-building system based on years of experience working with the moving industry. provides the only platform you'll ever need for driving traffic and quality online inquiries.
January 29, 2014 (Newswire.com) - Long-standing website development and SEO specialist Rank My Web is pleased to announce a new industry-specific lead generation program to its clients. This program, which can include everything from a brand new website, optimized content and an active search results improvement program or anything in between is aimed directly at the moving and relocation industry. Based in no small part on Rank my Web's more than 13 years of experience in working with the moving industry, this new lead generation program is custom-created to assist those companies looking to expand and prosper in today's market.
"The moving and relocation industry is a $25 Billion per year business," stated Uri Levy of Rank My Web, "and when you add transportation and storage, the figure grows even higher. Our goal is to help these businesses take advantage of what is available online today and prosper in an ever-changing business landscape."
In a study by WorldWide ERC an estimated $9.3 billion alone was spent in the U.S. in 2013 by companies relocating new and existing employees. When you consider that moving an employee homeowner costs between $72K and $92K and about a third of that for renters, and then factor in that relocating and tele-commuting continues to grow, it's clear that moving and relocation companies must do all they can to capture their market share.
For more detailed information please see the WorldWide ERc report at:
Rank my Web understands the needs and goals of the moving industry based on its many years of experience working closely with these companies. This new lead generation program is both all-encompassing and flexible. That means that Rank My Web can design a solution for any moving and relocation company. Whether it's a whole new system or fitting a program into the business's existing online and marketing resources.
"We not only know what our moving and relocation industry clients need and want," continued Levy, "but we are intimately familiar with the regulations that apply and know how to work within them for maximum effect."
Rank My Web pledges to ensure not only quality of service but strict adherence to all industry regulations. In this way, the company feels that it can best serve its new and existing clients and will continue to actively seek every opportunity to help them succeed.
About Rank My Web LLC
Rank My Web has quickly grown to be the only true choice for Full-Service Online/Digital Marketing and Advertising. Our development team and customer service agents, take pride in listening to your business needs and working with you to create a customized digital marketing plan that suits your needs and provides the only platform you'll ever need for driving traffic and quality online inquiries.
123 NW 13th St.
Boca Raton, FL 33432
Categories: Marketing
Tags: moving company, Moving Leads, moving Software, SEO Services, Web design


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BirminghamMargaret Wilson Scholarship recipients announced
The Margaret Wilson Scholarship recipients for 2014 are:
Courtney Kerr, daughter of Mickey and Jodi Kerr, who will attend University of Iowa;
Hanna Kerr, daughter of Mickey and Jodi Kerr, who will attend University of Northern Iowa;
Devon Ryan, daughter of Craig and Rhuie Ryan, who will attend University of Northern Iowa;
Jonathan Shaw, son of Morrie and Terry Shaw, who will attend University of Phoenix;
Dalton Stockwell, son of Adam and Melinda Stockwell, who will attend Indian Hills Community College; and
Dalton Stine, son of Phil Stine, who will attend Kirkwood Community College.
The recipients will be recognized during the 10 a.m. worship service May 19 at Birmingham United Methodist Church.
The Birmingham Lions will have a regular meeting at 7:30 p.m. Monday at the Lions Building. The committee is Van Meek and Harry Anderson.
The Over 50 and Fabulous Red Hat Ladies will meet for lunch at 11:30 a.m. Tuesday at Pizza Ranch in Fairfield. The hostesses are Joyce Dorothy and Arlene Lyon.
The Birmingham United Methodist Church board will meet at 7 p.m. Wednesday at the church.
— Arlene Lyon, Ledger correspondent


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My Nonprofit Reviews
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Review for Art Expression, Inc., Pittsburgh, PA, USA
I am an active nonprofit volunteer in the Greater Pittsburgh area. I have had first hand experience with the entire Art Expression community. They are a group of passionate, creative individuals committed to making a positive change in the lives of the students they serve.
More Feedback
Would you volunteer for this group again?
For the time you spent, how much of an impact did you feel your work or activity had?
Did the organization use your time wisely?
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Role: Volunteer


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The four players Sporting Kansas City (MLS) loaned to their affiliate Orlando City SC (USL PRO) will not be made available to play in the Lamar Hunt U.S. Open Cup fourth round match-up, based on an agreement between the two partnered clubs.
Midfielder Christian Duke, forward Dom Dwyer, goalkeeper Jon Kempin and defender Yann Songo'o have been with Orlando City SC since March 14th and helped them to the top of the USL PRO standings. The two-year partnership between the Lions and Sporting KC became official in January.
The upcoming game between Sporting Kansas City and Orlando City SC will kickoff at 7:30pm CT on Wednesday, June 12 at Sporting Park. The two teams met last year in the third round of the tournament, with Sporting Kansas City advancing 3-2 en route to winning their second Lamar Hunt U.S. Open Cup championship.


text: Hi guys, I have htc one x for over a year now. it was working fine but recently it just gone dead. i haven't done any firmware update or root device etc. i gave it to one repair shop the guy said that it might have memory IC problem and other things (processor, power ic) are fine but memory ic is not available right now. i showed it to another shop but that guy said that its processor is gone dead and there is nothing you can do. i don't know what to do. plz help me, if there is any possible solution for y device??


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Crawfordsville News
The Crawfordsville Lions Club met at 7 p.m. Monday, March 24, at the Crawfordsville Community Room. Twelve members were present.
President Larry Fletcher opened the meeting by leading the Pledge to the Flag and giving the opening prayer.
The minutes from the March 10 meeting were approved as read. The treasurer reported the balances in the regular and activity accounts.
Upcoming community events were discussed-the Easter Egg Hunt sponsored by the Crawfordsville Community Club on Saturday, April 5 at 10 a.m., as well as the July 5 Fun Day, which the Lions will co-sponsor.
The slate of officers were presented for balloting: president, Mindy Rochwick; first vice president, Loree Stodgill; 2nd Vice President: Billie Jo Rose; 3rd Vice President: Lance Miller; Secretary: Nancy Love; Treasurer: Randy Finke; Tail Twister: Larry Fletcher; Lion Tamer: Mike Massey; 1 Year Directors: Rick Fulton and Larry Swailes; 2 Year Directors: Craig Koehler and Gene Lintz
The vote was unanimous to accept these.
Road cleanup will take place April 5, beginning at 9 a.m. People are to meet at the town sign at the edge of town. Rain date is April 12. Randy Finke will take care of procuring the bags and recruiting Leo Club members to assist. Comments about the upcoming District Convention were exchanged. Larry Love mentioned he had attended the Wayland Lions Club meeting and the club, which the Crawfordsville Lions sponsored, would be celebrating a notable anniversary soon.
It was noted that the club will have the two WACO exchange students as speakers at the next dinner meeting, April 14
New member Adam Mitchell was installed into Lions by Arvid Anderson. The club is delighted to have him as a valuable addition to the club.
The meeting then adjourned.
Delicious refreshments were served by Randy Finke, and while everyone was enjoying them Tail Twister Larry Fletcher teased their brains with some trivia questions.
Holy Week will begin with Maundy Thursday services at the Ainsworth Community Church from 4 to 7 p.m. on April 17, Good Friday services are at The United Church of Crawfordsville on April 18. The United Church of Crawfordsville will have a Sunrise Service on Easter Sunday, April 20 at 7 a.m., with breakfast following.


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Goldmund: Famous Places
For all its spare elements and quiet stillness, Famous Places ends up delivering its own brand of expansiveness.
In his third release under the Goldmund moniker, composer Keith Kenniff gives us another striking set of solo piano pieces. As the album's title, Famous Places, implies, each of this record's 15 tracks represents a specific place, and each carves out its own landscape in doing so. These songs can be fluid lullabies, muted, plunking dystopias, spacious reflections, and whatever else they want to be. The negative space around Kenniff's notes is as much a vital sound here as the notes themselves, and he smartly helps the notes echo out in places with the faintest -- and I mean barely there -- ambient flourishes. Famous Places is an insistently quiet record, one that forces you to turn the stereo way up, or to cup your hands over your headphones to hear each aching note. However, that agency for the listener, that interaction you must have to fully absorb this music, is what makes it such a worthwhile experience. When you do your part, Kenniff rewards you with a rich and varied set, and one that, for its spare elements and quiet stillness, ends up delivering its own brand of expansiveness.


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I haven't been around a lot lately and I do apologize. I have been trying to take full advantage of summer. PROJECTS :: I have worked on hard on 2 outdoor projects, redoing my outdoor table and spray painting my plastic chairs. I finally got the projects done and was so excited since I had […]
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Promise rings are a beautiful reminder of a pledge. Whether your promise is to be true, to look forward to a future together, or to remain chaste, promise rings help you keep your word. Gemvara promise rings are made to order just for you in your choice of gems and precious metals to perfectly symbolize your pledge. Our wide selection of december birthstone tanzanite mothers promise jewelry means you can find a style that's perfect for you. At most retailers, mother's designs are limited and, frankly, dowdy. Gemvara believes that mother's jewelry should be as fashionable as everything else you wear. Our promise rings are crafted in the finest materials. Tanzanite is mined in only one place: near Kilimanjaro in Tanzania. Gemvara's tanzanite is a velvety blue with a hint of purple. Tanzanite is a December birthstone and the 24th anniversary gem. The December birthstones, blue topaz and tanzanite, have beautiful blue tones that you'll enjoy wearing with everything you own.
With a little care, promise rings will wear beautifully. Clean december birthstone tanzanite mothers promise jewelry in a bowl of water with a little mild dish soap. You can use a soft brush where dust collects. Gemvara's fine quality december birthstone tanzanite mothers promise jewelry are designed to be passed down for generations, hand-made by skilled craftspeople in the United States.
We can help right away. Chat with a jewelry expert now!


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Three years ago, [lastfm]Duran Duran[/lastfm] released Red Carpet Massacre, which ended up being one of the group's lowest-selling albums ever. For its 2010 comeback, though, they boys from Birmingham have lined up a super-producer to help.
Find out who it is after the jump.
[pullquote quote="Duran Duran is my first musical obsession." credit="Producer Mark Ronson"]
It's none other than [lastfm]Mark Ronson[/lastfm], who's produced Lily Allen & Amy Winehouse's wildly successful albums.
"[lastfm]Duran Duran[/lastfm] is my first musical obsession," said Ronson, who is best known as the producer behind chart-toppers like[lastfm]Lily Allen[/lastfm] and [lastfm]Amy Winehouse[/lastfm]. "There was a dude that cut the hair of all the kids at school, and I actually went with a picture of ([lastfm]Duran Duran[/lastfm] bassist) John Taylor in all his coiffed glory and had the nerve to ask for it like that. I love that band."
It's not the first time that the British pop superstars have worked with a man-of-the-moment producer; much of Red Carpet Massacre was produced by hitmaker [lastfm]Timbaland[/lastfm].
Meanwhile, Ronson loves horn sections, as his instrumental cover of [lastfm]Coldplay[/lastfm]'s "God Put a Smile On Your Face" from his solo album Version would indicate:
Do you think this is a match made in heaven? Or will it do nothing but remind people of just how good "Rio" is? Sound off below!
[Souce: The Guardian]


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Most of the talk about big data in government has been about crunching through existing data pools to spot trends that older tools weren't powerful enough to identify.
Computer scientists are crawling the full database of Medicare claims, for example, to spot common fraud patterns before those claims are paid out. IBM's Smarter Cities project is analyzing existing data from traffic sensors to build plans to ease congestion and reduce pollution.
The potential insights offered by deeper computer analysis have also prompted some agencies to begin new data gathering projects, though. Witness this special notice from the Centers for Disease Control and Prevention seeking a data coordinating center for information related to sudden death in people under 24 years old.
According to the notice:
Sudden death in the young (SDY) is a tragedy that affects children and young adults of all ages, making it a critical public health concern. Development of effective screening and prevention strategies is currently limited by the lack of prospectively defined epidemiological data, including incidence rates of specific causes of death (e.g., sudden cardiac death, sudden unexplained death in epilepsy). To address this knowledge gap, the CDC and the National Institutes of Health (NIH) are developing a program to explore and provide greater understanding of SDY by developing a surveillance system and registry that will broaden and enhance the activities of CDC's Sudden Unexpected Infant Death (SUID) Case Registry. We expect many of the infant SDY cases to be a subset of the SUID cases.
The National Institutes for Health is cosponsoring the project.
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Joseph Marks
Joseph Marks covers cybersecurity for Nextgov. He previously covered cybersecurity for Politico, intellectual property for Bloomberg BNA and federal litigation for Law360. He covered government technology for Nextgov during an earlier stint at the publication and began his career at Midwestern newspapers covering everything under the sun. He holds a bachelor's degree in English from the University of Wisconsin in Madison and a master's in international affairs from Georgetown University.