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Better, worse or the same: Falcons special teams
Special teams are fundamental to a team's success. Atlanta's special teams efforts and roster have been pretty consistent over the past few seasons. What's the outlook for this group this year? Current Special Teams Roster Punter Matt Bosher Kickers Matt BryantNick Rose Long snapper Josh Harris Potential return specialists Devin HesterEric WeemsDevin FullerJustin HardyNick...
Via The Falcoholic
14 hours ago
Grading Falcons GM Thomas Dimitroff: 2014 free agency
Where free agents are concerned, this class wasn't awful. We continue our review of Thomas Dimitroff's time as GM of the Falcons. Today, we take a look at 2014 free agency. This particular period was critical for the team, as the team was coming off a dismal 4-12 season and both Mike Smith and Thomas Dimitroff were considered to be on the hot seat. Hitting big in both the draft...
Via The Falcoholic
18 hours ago
Falcons training camp is six days away. Here's six remaining roster holes for Atlanta
The Falcons are an improved team, but they still have some question marks that could be an issue in 2016. Less than a week until training camp for the Atlanta Falcons, and we're counting down every boring, stupid day between now and then. Six to go! Today, we're going to look at the remaining roster holes the Falcons have coming into training camp, holes that might be addressed...
Via The Falcoholic
22 hours ago
Falcons have reinforcements for second camp under Dan Quinn
FLOWERY BRANCH, Ga. (AP) The Atlanta Falcons are hoping their second season with coach Dan Quinn and offensive coordinator Kyle Shanahan brings more familiarity, confidence - and wins. A few new key players might make an even bigger difference. The Falcons improved from 6-10 to 8-8 but missed the playoffs in Quinn's first season. There was improvement, especially on defense, but...
Via Associated Press
1 day ago | Discuss
Atlanta Falcons capsule
ATLANTA FALCONS (8-8) OPEN CAMP: July 28, Flowery Branch, Georgia LAST YEAR: There was great buzz when Falcons started 5-0 under first-year coach Dan Quinn, but team couldn't overcome 30 turnovers as it lost seven of eight, including six straight, and missed playoffs. Quarterback Matt Ryan's production declined in first year with offensive coordinator Kyle Shanahan; 21 TD passes...
Via Associated Press
1 day ago | Discuss
TGS 2016 Preview: Atlanta Falcons
Between now and the end of August, TGS will offer up a quick snapshot of every NFL team heading into the 2016 regular season. Today, the Atlanta Falcons. 2015 in a nutshell: Despite starting 5-0 and possessing a ton of talent, the Falcons finished 8-8 in their first season under Dan Quinn, failing to make the playoffs for the third consecutive year. Why 2016 could be different: Year...
Via This Given Sunday
2 days ago | Discuss
Falcons Reach Injury Settlement With C Stone
Atlanta has reached an injury settlement with center James Stone. The Falcons' roster now sits at 88 men, leaving two roster spots open prior to the start of Training Camp.
Via AtlantaFalcons.com
3 days ago
Rich McKay on Marion Campbell (1929-2016)
Atlanta Falcons team president Rich McKay. (John Raoux/Associated Press) Marion Campbell, the only man to serve two stints as the head coach of the Atlanta Falcons, died last week in Plano, Texas, it was announced late Sunday. He was 87. Campbell was a three-time All-SEC pick at Georgia from 1949-51 and played eight seasons in the NFL as one of the last two-way players before beginning...
Via AJC's Atlanta Falcons Blog
3 days ago
Frank Kleha on Marion Campbell (1929-2016
Former Falcons executive Frank Kleha (right) being interviewed. Marion Campbell, the only man to serve two stints as the head coach of the Atlanta Falcons, died last week in Plano, Texas, it was announced late Sunday. He was 87. Campbell was a three-time All-SEC pick at Georgia from 1949-51 and played eight seasons in the NFL as one of the last two-way players before beginning his...
Via AJC's Atlanta Falcons Blog
3 days ago
Matt Ryan: Deflategate will not affect Brady's legacy
Whether he deserves it or not, Tom Brady is going to have a four-game suspension on his resume when his playing career comes to an end. Will that negatively impact his legacy? Matt Ryan doesn't think so. TMZ recently caught up with Ryan to get his opinion on Deflategate. When asked specifically if it puts a stain on what Brady has accomplished, Ryan defended the Patriots quarterback...
Via Larry Brown Sports
4 days ago | Discuss
Who knew Matt Ryan was actually funny?
The offseason has been very informative for Atlanta Falcons fans when it comes to Matt Ryan. After coming off as a pretty boring, bland dude for the first eight years of his career with the Falcons, he's finally showing some of his personality on his Twitter feed this offseason. It all started in May with this gem: What Julio sees when I throw him a go route #RiseUp pic.twitter...
Via Sportsnaut
9 days ago | Discuss
Atlanta Falcons: 2016 the year of Matt Ryan?
When Matt Ryan left Boston College and was drafted 3rd overall in the 2008 NFL Draft, the Atlanta Falcons knew they would have a franchise quarterback for at least a decade. Fortunately for the Falcons, a five-year extension signed in 2013 will keep him with the team until the end of the 2018 season. His first five years with the team brought some of the most unprecedented numbers...
Via isportsweb.com
10 days ago | Discuss
Falcons want to reduce Devonta Freeman's workload
As a sophomore, Falcons running back Devonta Freeman ranked fourth in the NFL with 264 carries last season. Now the team is working toward easing off the gas pedal heading into 2016. The Falcons are looking to reduce Freeman's workload next season, according to running backs coach Bobby Turner. "It's about growth," Turner said to D. Orlando Ledbetter of the Atlanta Journal...
Via Larry Brown Sports
11 days ago | Discuss
Atlanta Falcons NFL Season Preview
NFL Season Preview for the Atlanta Falcons.
Via Walter Football
14 days ago | Discuss
Roddy White was 'miserable' with the Falcons last season
Former first-round pick Roddy White spent 11 seasons with the Atlanta Falcons before his release following the 2015 season. While the four-time Pro Bowl receiver had pleasant memories during his long stint in Atlanta, he recently discussed the major disconnect he experienced with the team this past season. "Being released wasn't a bad thing for me, because I wasn't going...
Via Sportsnaut
15 days ago | Discuss
Atlanta Falcons: Justin Hardy Spotlight
Justin Hardy has the skills to be an effective wide receiver in the Atlanta Falcons offensive scheme. The 5'10" receiver was drafted in the fourth round of the 2015 NFL Draft out of East Carolina University. Hardy had a very successful college career. He finished his college career with 387 receptions for 4,541 yards and 35 touchdowns. He is the all-time leading NCAA receptions...
Via isportsweb.com
16 days ago | Discuss
Falcons DC says Keanu Neal will be 'pretty special'
The Atlanta Falcons had many options to choose from with the 17th overall pick in the 2016 NFL Draft, ultimately selecting Florida safety Keanu Neal. So far, it looks like the coaches in Atlanta are impressed by the player and believe he could be a big part of their defense going forward. Defensive coordinator Richard Smith recently spoke with the team's official website and talked...
Via Campus Sports Net
17 days ago | Discuss
Dan Quinn doesn't believe in two-point conversions
It's been a debate that has picked up steam since the rule was changed last season, where the extra-point kick attempts were moved to the 15-yard line to encourage teams to go for two. Atlanta Falcons head coach Dan Quinn doesn't feel the needed to go for two still and doesn't like the idea of doing it every single time, as he told the Atlanta Journal-Constitution: �...
Via The Comeback
25 days ago | Discuss
Atlanta Falcons: Devonta Freeman, boom or bust?
Devonta Freeman had himself quite a year during the 2015 regular season. He was instrumental with the early success of the team. In his rookie year, the 2014 regular season, he had just 248 rushing yards and an additional 225 receiving yards. To go along with the almost 500 all-purpose yards, he had two touchdowns. Moving to his sophomore year, the 2015 regular season was much...
Via isportsweb.com
June 22, 2016 | Discuss
Falcons GM Thomas Dimitroff knows he's on hot seat
Falcons general manager Thomas Dimitroff is on the hot seat entering the 2016 season, and he'll be the first to tell you. "Of course I'm on the hot seat. Every year I'm on the hot seat," Dimitroff told the Talk of Fame Network. Dimitroff has been the Falcons' GM for nine years and acknowledges how fortunate he is to be in that position for that long at age 49. While...
Via The Sports Daily
June 22, 2016 | Discuss
Atlanta Falcons cut concession prices to lowest in all major pro sports
If you're an Atlanta Falcons fan looking to get some popcorn and a hot dog at the new Mercedes-Benz Stadium, you won't have to spend a small fortune to get it. While stadium prices are notoriously high, Falcons owner Arthur Blank announced on Monday that prices at the team's new stadium will be the lowest in all of the four major professional sports. The prices are legit....
Via The Comeback
Falcons are 'thanking fans' with industry low concession prices at Mercedes-Benz Stadium
The Atlanta Falcons are thanking their fans by offering the lowest concession prices among all major American sports, starting in 2017 with the opening of the franchise's brand new Mercedes-Benz Stadium. The Falcons will be offering non-alcoholic beverages (plus free refills), Dasani bottled water, hot dogs, pretzels and popcorn for just $2 each, and peanuts, pizza, nachos and...
Via The Sports Daily
Falcons' new stadium to have fan-friendly concession prices
For most NFL fans, in most cities, buying concessions at the game involves some combination of arm and leg removal. The Atlanta Falcons are doing what they can do change the narrative on how much it costs to eat and drink while enjoying the game at their new stadium. Mercedes-Benz Stadium, set to open in time for the 2017 NFL season, will almost surely feature the most affordable...
Via Sportsnaut
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Ivanka Trump defends father during CNN interview segment against media!
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Ready in 1 hour
If you are making the pie right away (not freezing the filling in order to use later), just mix the filling ingredients and put into the crust (no need to let it stand or to cook on the stove).
81 people want to try | 206 have favorited
Peel, core and thinly slice the apples. In a large bowl, toss with the other ingredients. Let stand for 30 minutes. Cook over medium heat till mixture begins to thicken. Pour into 1/2-gallon size freezer bags. Seal, label and freeze.
To make a pie:
Thaw a bag of filling. Heat oven to 425?. Make a 2-crust piecrust following the directions on the Crisco can. Put filling into the crust and dot with 1 Tbsp butter, cut into bits. Bake 40-50 mins till golden.
More ideas
rebekahbalamchuc 2y agoI substitutes gf multi purpose flour and it was great!!
CookByMarriage 5y agoGreat recipe. We pick apples in the fall and prepare a bunch of bags of filling, labeling each bag with cooking instructions. Then it's a no brainer to pop it in the oven - or to give it away. Nothing to look up!
free82 6y agoGreat idea love the simplicity Maryann from niles ohio
bkprice13 8y ago[I posted this recipe.]


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10th Annual Peabody Charter School 5K/10K
Goleta Beach Park
Sun 03/02/2014
These are a Grand Prix 10K (at 8am) and 5K (at 9:15am) which will start and finish at Goleta Beach. Both events will run out along the bike path and Atascadero Creek. The 10K will turnaround just before Puente. The 5K turnaround is just before Patterson. Both the turnarounds will include a water station. Children are welcome and encouraged to run EITHER or BOTH events. However, the 5K will probably be more geared toward being a family fun run. NOTE: Elementary school kids, if pre-registered by picking up an application at Peabody Charter School (or emailing the race director) run FOR FREE. Race day fee is $5.Event schedule and timesGoleta Beach Park, Area D: 10K starts at 8am sharp on March 2, 2014 5K starts at 9:15am on March 2, 2014
- Address:5986 Sandspit Rd
Goleta, CA 93117


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This paper presents methods of modeling and predicting face recognition (FR) system performance based on analysis of similarity scores. We define the performance of an FR system as its recognition accuracy, and consider the intrinsic and extrinsic factors affecting its performance. The intrinsic factors of an FR system include the gallery images, the FR algorithm, and the tuning parameters. The extrinsic factors include mainly query image conditions. For performance modeling, we propose the concept of "perfect recognition", based on which a performance metric is extracted from perfect recognition similarity scores (PRSS) to relate the performance of an FR system to its intrinsic factors. The PRSS performance metric allows tuning FR algorithm parameters offline for near optimal performance. In addition, the performance metric extracted from query images is used to adjust face alignment parameters online for improved performance. For online prediction of the performance of an FR system on query images, features are extracted from the actual recognition similarity scores and their corresponding PRSS. Using such features, we can predict online if an individual query image can be correctly matched by the FR system, based on which we can reduce the incorrect match rates. Experimental results demonstrate that the performance of an FR system can be significantly improved using the presented methods
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text:
|Publication number
|Publication type
|Application number
|Publication date
|Mar 24, 1998
|Filing date
|Jun 7, 1995
|Priority date
|Jul 12, 1993
|Fee status
|Also published as
|Publication number
|Oleg Voloshin, Lijiang Wang, R. Daniel Camerini-Otero
|Original Assignee
|The United States Of America As Represented By The Department Of Health And Human Services
|Export Citation
|BiBTeX, EndNote, RefMan
|Patent Citations (3), Non-Patent Citations (40), Referenced by (17), Classifications (7), Legal Events (6)
|External Links: USPTO, USPTO Assignment, Espacenet
This application is continuation-in-part of U.S. application Ser. No. 08/089,910, filed Jul. 12, 1993, now U.S. Pat. No. 5,460,941; and is also a continuation-in-part of U.S. application Ser. No. 08/097,831, filed Jul. 26, 1993, now 5,510,473. The complete disclosures of both of these related applications is hereby incorporated herein by this reference thereto.
The present invention relates to peptides capable of promoting homologous DNA pairing and the use of the compositions containing the peptides in promoting such DNA pairing. More specifically, the invention relates to short peptides derived from the E. coli recA protein which are capable of catalyzing homologous DNA pairing.
RecA proteins play an essential role in homologous DNA recombination pathways (Cox et al., Bioessays, 15:617-623, 1993; Kowalczykowski et al., Ann. Rev. Biochem., 63:991-1044, 1994). The first RecA protein was purified from E. coli in 1965 by Clark et al. (Proc. Natl. Acad. Sci. USA, 53:451, 1965) and since then has been isolated and cloned from many other prokaryotic organisms (Roca et al., Biochem. Mol. Biol., 25:415-456, 1990). The RecA protein is a 38 kD polypeptide which possesses a multitude of biochemical activities.
RecA promotes homologous DNA recombination via a multi-step pathway involving the formation of a single-stranded nucleoprotein filament, DNA pairing and subsequent strand exchange to form heteroduplex DNA. RecA is directly involved in post-replication DNA repair and the induction of the SOS response by cleavage of the lexA repressor protein. RecA mediates a set of DNA strand exchange reactions in vitro by self-assembly into filaments, binding to both single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA), and hydrolysis of ATP. Although the crystal structure of the protein has been solved (Story et al., Nature, 355:318-325, 1992), the precise domain(s) which catalyze the pairing of two homologous DNA molecules are unknown. RecA binds to ssDNA, forming a nucleoprotein (presynaptic) filament that is the homology searching moiety that mediates the pairing with a target DNA duplex.
One embodiment of the present invention is a peptide comprising at least 15 consecutive amino acids of the amino acid sequence shown in SEQ ID NO:1 or conservative amino acid substitutions thereof, the peptide being capable of promoting pairing of a single-stranded DNA molecule and a double-stranded DNA molecule, wherein the single-stranded DNA molecule is homologous to at least a portion of the double-stranded DNA molecule. Preferably, the peptide has the amino acid sequence shown in SEQ ID NO:1. Alternatively, the peptide has an amino acid sequence shown in SEQ ID NO:2 or SEQ ID NO:3.
Another embodiment of the invention is a method for selectively targeting a single-stranded DNA molecule to a double-stranded DNA molecule containing at least a portion of a sequence homologous to the single-stranded DNA molecule, comprising contacting the single-stranded DNA molecule and the double-stranded DNA molecule in the presence of a peptide described above. Advantageously, this peptide has the amino acid sequence shown in SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3. According to another aspect of this preferred embodiment, the targeted DNA region of the double-stranded DNA molecule encodes a mutant form of a protein.
The present invention also provides a method of inhibiting transcription of a specific gene sequence present on one strand of a double-stranded DNA molecule comprising contacting a peptide with the double-stranded DNA molecule and with a single-stranded DNA molecule, which single-stranded DNA molecule is homologous to at least a portion of the gene sequence and hybridizes therewith to form a three-stranded molecule, wherein the peptide is a peptide described above. Preferably, the peptide has the amino acid sequence shown in SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3. Advantageously, the specific gene sequence on the double-stranded DNA molecule encodes a mutant form of a protein. Alternatively, the specific gene sequence on said double-stranded DNA molecule encodes an oncogene or viral transcript.
FIG. 1a illustrates the peptides used in DNA binding experiments. FECO is the naturally-occurring sequence of residues 193-212 of E. coli RecA (SEQ ID NO:1). WECO (SEQ ID NO:2), YECO (SEQ ID NO:3), HECO (SEQ ID NO:4) and AECO (SEQ ID NO:5) contain, in place of phenylalanine at position 203, tryptophan, tyrosine, histidine and alanine, respectively. WT-14 is the naturally occurring sequence of residues 196-209 of RecA (amino acids 4-17 of SEQ ID NO:1). WT-Scr (SEQ ID NO:6) is a randomly scrambled sequence having the same amino acid composition as FECO.
FIG. 1b illustrates binding of the FECO, WECO, YECO and AECO to ssDNA (0.5 μM) in the presence of 10 mM MgCl2. The peptide concentration is shown on the x-axis and the percentage of ss DNA bound is shown on the y-axis.
FIG. 1c illustrates binding of the FECO, WECO, YECO and AECO to dsDNA (1.0 μM) in the presence of 10 mM MgCl2. The peptide concentration is shown on the x-axis and the percentage of dsDNA bound is shown on the y-axis.
FIG. 1d illustrates binding of the FECO, WECO and YECO to ssDNA (0.5 μM) in the absence of MgCl2. The peptide concentration is shown on the x-axis and the percentage of ssDNA bound is shown on the y-axis.
FIG. 1e illustrates binding of the FECO, WECO and YECO to dsDNA (1.0 μM) in the absence of MgCl2. The peptide concentration is shown on the x-axis and the percentage of dsDNA bound is shown on the y-axis.
FIG. 2a illustrates a circular dichroism (CD) spectrum of a reaction mixture in which a 57-mer ssDNA molecule (CTGTCTACTCTCGAGGTTAACCCGTGCGAATTCTACGATTGGTGCGGCCGGT ATATC; SEQ ID NO:7; 0.3 mM) was incubated at pH 7.5�0.2 in the presence of 0.1 mM FECO (◯) or with FECO at concentrations of 0.20 mM (⋄), 0.25 mM (□) and 0.40 mM (Δ).
FIG. 2b illustrates a CD spectrum of 0.10 mM WECO in the presence or absence of 0.20 mM ssDNA (SEQ ID NO:7) at pH 6.0�0.2. The inset shows the enlargement of the spectral region from 260-300 nm to emphasize the intensity change at 278 nm.
FIG. 2c illustrates a CD spectrum of 0.10 mM WECO in the presence (◯) or absence (⋄) of 0.40 mM dsDNA from the HaeIII digestion products of plasmid pUC18 at pH 6.0�0.2. The inset shows the enlargement of the spectral region from 260-300 nm to emphasize lack of the intensity change at 278 nm.
FIG. 2d illustrates a CD spectrum of 0.40 mM HECO in the presence (◯) and absence (⋄) of 0.4 mM ssDNA at pH 7.5�0.2.
The sequence-specific targeting of DNA by RecA has important therapeutic applications including site-specific gene inactivation, correction of gene mutations and the control of gene expression. Other applications include sequence-specific mapping and manipulation of complex genomes, particularly the human genome.
Sequence-specific targeting of DNA is described in copending U.S. application Ser. No. 08/089,910. This copending application describes contacting ds DNA with a ss oligonucleotide probe complementary to a specific sequence in the presence of the entire E. coli RecA protein, resulting in triplex formation in vitro.
However, the use of whole, intact RecA protein for specific targeting of DNA presents several drawbacks typically associated with whole proteins. These drawbacks include the difficulty of whole protein uptake by cells, delivery of the protein in sufficient concentration to cells to mediate an effect, generation of a significant antibody response, susceptibility to biodegradation and the difficulty in modifying large proteins to increase their bioactivity. In addition, the production of recombinant protein is labor-intensive and expensive in comparison to the synthesis of small peptides.
Thus, there is a need for a smaller, more stable molecule capable of being easily introduced into cells both in vitro and in vivo for targeting ds DNA in the genome, both for therapeutic and mapping/cloning applications. The present invention addresses this need.
The present invention relates to the discovery that a twenty amino acid peptide spanning the ssDNA binding domain of the homologous recombination-promoting RecA protein binds to both ssDNA and dsDNA, promotes unstacking of the DNA base pairs, and, most importantly, selectively catalyzes the formation of joint ssDNA-dsDNA molecules at a region of homology between the ssDNA and dsDNA. The single-stranded DNA binding domain of E. coli RecA is in a disordered mobile loop in the crystal structure (Gardner et al., Eur. J. Biochem., 1995 (in press). It is quite unexpected that such a short peptide is capable of carrying out the most unique and characteristic reaction mediated by the whole RecA protein: pairing of homologous single- and double-stranded DNA molecules and the formation of joint molecules. It is also quite surprising that these short peptides can mediate such complex reactions, as the native RecA protein has a molecular weight of about 20 times that of the peptide and it would be expected that additional regions of the protein beyond the 20 amino acids would be required for its activity.
The short peptides of the present invention have the sequence of the E. coli single strand binding domain, or sequence variations thereof. As explained in detail hereinbelow, these peptides can selectively target a single-stranded oligonucleotide to a homologous sequence contained in a double-stranded DNA molecule. This selective targeting has therapeutic applications, including inactivation of deleterious genes and control of gene expression, and may be used to map complex genomes, including the human genome.
The 20 amino acid peptide capable of promoting homologous DNA pairing corresponds to amino acids 193-212 of native RecA (SEQ ID NO:1) and variations thereof that retain activity. A variety of such variations on the 20-mer can be made. The synthetic peptides and oligonucleotides described herein were made according to the techniques described below in Example 1.
Oligonucleotides were synthesized on an Applied Biosystems model 380B synthesizer and purified by polyacrylamide gel electrophoresis (PAGE). The peptides used in the examples herein were made on an Applied Biosystems model 431A synthesizer, purified by reverse phase high performance liquid chromatography (HPLC) on a C-18 column and dissolved in 20 mM CAPS (3- Cyclohexylamino!-1-propane-sulfonic acid; Sigma, St. Louis, Mo.), pH 10.6.
The 14-mer, WT-14, described above in the brief description of FIG. 1a, lacks the first three and last three amino acids of the active 20-mer. This 14-mer did not promote the formation of joint DNA molecules. Thus, RecA-derived peptides having 15 amino acids or more, and including amino acids 196-209 (the 14-mer), are within the scope of the invention. Although longer peptides are included within the scope of the present invention, the preferred upper limit of the length of the peptide would be about 50 amino acids, i.e. the 20-mer plus 30 additional residues of native RecA in combination on the N-terminal and/or C-terminal sides of the 20-mer. Peptides longer than about 50 amino acids would be more difficult to manipulate, more difficult to introduce into cells and subject to more of the drawbacks described above in connection with whole proteins, including degradation and immunological intolerance.
It will be appreciated that 20 amino acid peptides containing one or more amino acid substitutions in various positions of the sequence shown in SEQ ID NO:1 are also within the scope of the invention. Many amino acid substitutions can be made to the native 20-mer sequence and retain the activity of the peptide. However, in the preferred embodiment of the invention, the asparagine, glutamine, glycine and glycine residues at positions 193, 194, 211, and 212 of native RecA, respectively, are present in the corresponding positions of the peptides of the invention (positions 1, 2, 19 and 20), as these residues are highly conserved among RecA-like proteins. Moreover, the presence of the aromatic amino acids phenylalanine, tyrosine or tryptophan at position 203 is also preferred.
Variations of SEQ ID NO: 1 contemplated for use in the present invention include minor insertions, deletions or substitutions that do not substantially affect its ability to bind and unstack DNA and to catalyze the formation of joint ssDNA-dsDNA molecules. For example, conservative amino acid replacements are contemplated. Such replacements are, for example, those that take place within a family of amino acids that are related in their side chains. The families of amino acids include the acidic amino acids (aspartic acid, glutamic acid); the basic amino acids (lysine, arginine, histidine); the non-polar amino acids (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and the uncharged polar amino acids (glycine, asparagine, glutamine, cysteine, serine threonine, tyrosine); and the aromatic amino acids (phenylalanine, tryptophan and tyrosine). In particular, it is generally accepted that conservative amino acid replacements consisting of an isolated replacement of a leucine with an isoleucine or valine, or an aspartic acid with a glutamic acid, or a threonine with a serine, or a similar conservative replacement of an amino acid with a structurally related amino acid, in an area outside of the polypeptide's active site will not have a major effect on the properties of the polypeptide.
In fact, any peptide derivative of SEQ ID NO:1, including conservative substitutions, non-conservative substitutions, mixtures thereof, as well as truncated peptides and longer peptide sequences containing additional amino acids of native RecA or sequence variations thereof may be tested as described in the examples set forth hereinbelow to determine their ability to bind ssDNA/dsDNA, unstack ssDNA and catalyze joint ssDNA-dsDNA molecule formation. Such routine experimentation will allow the skilled artisan to easily screen any desired peptide.
The peptides of the invention bind to both ssDNA and dsDNA in vitro, both in the presence and absence of magnesium ion as evidenced by a filter binding assay, as described below in Example 2.
dsDNA was prepared by annealing of 5' 32 P labeled oligonucleotide BS-S1 (5'-GGCCGCTCTAGAACTAGTGGAT CCCCCGGGCTGCAGGAATTCGATATCA AGCT-3'; SEQ ID NO:8) and unlabeled BS-S2 (5'-AGCTTGATATCGAATTCCTGCAGCCCGGGGATCCACTAGTTCTAGAGCGG CC-3'; SEQ ID NO:9). BS-S1 spans positions 742-690 in the polylinker region of the plasmid Bluescript SK+ (Stratagene, La Jolla, Calif.) and BS-S2 is the complement of BS-S1. Binding Reactions were performed in a volume of 40 μl and contained 32 P-labeled 0.5 μm ssDNA or 1 μm dsDNA (expressed as phosphate concentration), 40 mM Tris-borate, pH 7.5, 0 or 10 mM MgCl2, 20 mM NaCl, 10 μg/ml BSA, 10 mM CAPS, pH 10.6 and peptide concentrations of up to 100 μm; final pH value was 8.3. After a 30 minute incubation at room temperature, reaction mixtures were filtered using a double-filter system (Wong et al., Proc. Natl. Acad. Sci. USA, 90:5428-5432, 1993) with BA85 nitrocellulose filters and NA45 DEAE membranes (Schleicher and Schuell, Keene, N.H.). All experiments were performed in at least triplicate. Data were quantitated with a phosphorimager (Molecular Dynamics).
The results shown in FIGS. 1b and 1c indicate that an aromatic amino acid (phenylalanine, tyrosine or tryptophan) is needed at amino acid position 203 of RecA (amino acid 11 of SEQ ID NOS: 1-4) for binding to both ssDNA and dsDNA. The sequence shown in SEQ ID NO:4 in which residue 11 is an alanine did not bind to either ssDNA or dsDNA (FIG. 1b, c). Further, HECO, WT-14 and WT-Scr did not bind to either ssDNA or dsDNA. WT-Scr contains the same amino acid composition as FECO, but in a scrambled sequence. The phenylalanine residue at position 203 is the most conserved internal position among prokaryotic RecA proteins and their eukaryotic homologs, such as Dmc1 and Rad51 (Story et al., Science, 259:1892-1896, 1993). Although the peptides bind less tightly in the absence of magnesium ions than in their presence (FIGS. 1d, e), the fact that 100-200 mM NaCl can substitute for 10 mM Mg2+ indicates that this is not a specific divalent ion effect. As expected, ATP, ADP and ATPγS had no effect on binding of the peptides to DNA.
The peptides promote unstacking of DNA as assessed by increased sensitivity of thymine residues to the modifying reagent potassium permanganate. The ability of the aromatic amino acid-containing peptides to unstack DNA was addressed as described in the following example.
RecA is known to extend both ssDNA and dsDNA by 50% (Koller et al., in Mechanisms of DNA Replication and Recombination, N. Cozzarelli, Ed., Alan R. Liss, Inc., New York, 723-729, 1983). The unstacking of the ssDNA is proposed to be essential for RecA-mediated DNA triplex formation between ssDNA and ds DNA (Camerini-Otero et al., Cell, 73:217-223, 1993). Unstacked bases are more accessible to modification by potassium permanganate (PP), an agent that attacks thymines in a direction perpendicular to the base plane (Hayatsu et al., Biochem. Biophys. Res. Commun., 29:556-561, 1967). This modification is monitored by chemically-induced strand cleavage at the modified base (Maxam et al., Meth. Enzymol., 65:499-560, 1980).
Peptide-DNA complexes were prepared as described in Example 2, in a total volume of either 100 or 140 μl. In the latter case, 40 μl was used for filter binding and the remainder treated with PP. To bind RecA protein to BS-S1, 2.7 μm RecA and 0.5 μm 32 P-labeled BS-S1 were incubated in a volume of 100 μl containing 25 mM Tris-Hcl, pH 7.5, 10 mM MgCl2, 20 mM NaCl, 0.4 mM dithiothreitol (DTT), 0.5 mM EDTA, 0.3 mM ATPγS, 1.1 mM ADP and 10 μg/ml BSA for 30 min. at 37� C. Complexes were incubated with 0.5 mM PP for 1 min. at room temperature. Reactions were terminated with DMS-stop solution (Maxam et al., ibid.) and precipitated with ethanol. After treatment with 1M pyrollidone (95� C., 20 min.), the samples were separated on a polyacrylamide gel containing 20% urea (Sequagel, National Diagnostic). The gel was dried and exposed to x-ray film.
As expected, the ss oligonucleotide was much more reactive to PP in the presence of RecA than in its absence, as illustrated by the higher intensities of the bands. Peptides FECO and WECO induced pronounced hypermodification of thymine residues in the ssDNA. As a result, little if any full size fragments remain and most of the radioactivity is found in very short oligonucleotides, suggesting multiple hits by PP. The reactivity of the ssDNA to PP was also increased in the presence of YECO, but to a lesser extent than FECO and WECO. The shuffled non-DNA-binding WT-Scr peptide had no effect. These results indicate that, as with intact RecA, WECO and FECO unstack ssDNA upon binding thereto.
The extent of modification of the thymine residues in the ssDNA increased with FECO concentration in a manner which paralleled the binding profile. Interestingly, although the binding of FECO to dsDNA was similar to that for ssDNA, no distinct modification of dsDNA was observed using peptide concentrations between 0 and 25 μm. This suggests that the peptide uses different modes in binding to ssDNA versus dsDNA and that binding to dsDNA does not occur at single-stranded regions in the duplex.
The structure of the peptides themselves is changed upon binding to DNA as assessed by circular dichroism spectroscopy. In the absence of DNA, the peptides assume a random-coil conformation. However, upon DNA binding, the peptides shift from random-coil structure to β-sheet. This conformational change appears important in binding to ssDNA and dsDNA and unstacking the ssDNA.
Because the structure of the DNA changed upon peptide binding, the ability of the peptides to change their secondary structure (conformation) upon binding to DNA was investigated using CD spectroscopy as described below.
CD spectroscopy measures protein and nucleic acid secondary structure. All CD spectra were measured using a Jasco 720 spectropolarimeter. The instrument was calibrated with a 0.06% ammonium (+)-d10 -camphorsulphate solution which generated a CD intensity of 190.4 millidegrees (mdeg) at 290.4 nm. A baseline of 10 mM NaH2 PO4 buffer was subtracted out as background. Cells of 0.20, 0.50 or 1.00 mm pathlength were used and maintained at 22�1� C. The response and band width for data collection were 2.0 sec and 1.0 nm, respectively. Spectra were smoothed using Savitsky Golay smooth and are presented at 2 nm interval and in molar residue ellipticity (degree* M-1* cm-1) or as raw data (mdeg).
As shown in FIG. 2a, binding of FECO to ssDNA induced a conformational transition from random coil to mostly β-structure. While the dominant negative CD peak at approximately 198 nm at low peptide concentration indicated a random-coil structure, the amplitude increase at both 190 nm and 215 nm at higher peptide concentrations is characteristic of a concentration-dependent binding of a structure with a high β-structure content on the DNA (Woody et al., in The Peptides, Analysis, Synthesis, Biology, V. Hruby, Ed., Academic Press, Inc., New York, 1985, pp. 15-114). The peptide remains a random coil in the absence of DNA, even at higher peptide concentrations. The isodichroic point of 207 nm indicates that the data can best be described as a transition between two, and not more, states.
A similar structural change is also evident in WECO (FIG. 2b). However, WECO forms a more complete β-structure than FECO under the same conditions, most likely due to its higher affinity for DNA. All other peptides tested remained predominantly random coil structures in the presence of ssDNA, as typified by the HECO CD profile (FIG. 2d). In addition, the reduction in the 278 nm DNA band for the WECO-ssDNA complex (FIG. 2b, inset) supports the chemical modification data showing that the ssDNA is unstacked upon complex formation. Binding to dsDNA also induces β structure in WECO but, consistent with the chemical modification data, results in only minor changes in the 278 nm band (FIG. 2c).
The CD changes correlate well with the filter binding and chemical modification data, suggesting that CD and these methods are complementary and that the conformational change of the peptide from random coil to β structure is important in binding to ssDNA and dsDNA and unstacking the ssDNA.
Most importantly, the peptides of the invention promote joint molecule formation between ssDNA and dsDNA molecules having one or more regions of homology. The formation of joint molecules by the peptides does not require an external energy source, as nucleotide cofactors are unnecessary. This has also been demonstrated for RecA. It is contemplated that this joint molecule formation results in homologous DNA recombination and subsequent heteroduplex formation.
The ability of the peptides of the invention to promote homologous pairing of DNA molecules will allow introduction of a nucleic acid sequence into a precise location in the human genome. In a preferred embodiment, the peptide and a ssDNA sequence having homology to a particular sequence contained within the genome are introduced into particular cells in vivo. This may be accomplished, for example, by encapsulating the peptide and ssDNA in a liposome followed by injection into the bloodstream. The liposomes will fuse with cells in vivo, resulting in intracellular delivery of peptide and ssDNA sequence. The ssDNA sequence will then pair with its corresponding genomic sequence, forming a ssDNA-dsDNA three-stranded molecule. Alternatively, the peptide and ssDNA sequence may be directly coinjected. Because the peptide is small, there is reduced risk of a substantial immunological response compared to a large protein such as RecA which would elicit a substantial immune response. In addition, the peptide may be chemically modified by well known techniques to increase its stability.
RecA can bring together any two homologous DNA molecules and promote their recombination. Although FECO and WECO both bind ssDNA and dsDNA, assume a well-defined structure as a result of this binding, and unstack ssDNA, it would be unexpected that such short 20 amino acid peptides would selectively pair a ssDNA with a homologous dsDNA. This possibility was addressed in the following example.
The formation of joint (ssDNA-dsDNA) molecules was investigated by incubating 300 ng pBluescript KS+ (supercoiled plasmid) and 13 ng 32 P-BS-S1 with either 13.6 μm RecA or 50 μm peptide under conditions used for RecA and peptide-ssDNA formation described in Example 3, with the exception that reactions involving peptides were allowed to proceed for 90 min. at 45� C. The ratio between ss oligonucleotides and the target ds DNA sequence was 9-10 oligonucleotides per dsDNA molecule for both the peptide and RecA reactions. Reactions were quenched by the addition of 2% sodium dodecyl sulfate (SDS) and 20 mM EDTA, followed by electrophoresis on a 1% agarose gel containing 6 mM magnesium acetate and 0.5 μg/ml ethidium bromide for 4 hours in a cold room. The gel was dried and exposed to x-ray film.
Surprisingly, FECO and WECO catalyzed the formation of stable joint ssDNA-dsDNA (BS-S1/pBluescript) molecules similar to those formed by RecA. These joint molecules are detected as comigration of radiolabel with the plasmid on the agarose gel. We were able to recover from 10% to 20% as many joint molecules from reactions with these two peptides compared to the whole RecA protein. No joint molecules were formed when BS-S1 was replaced with a duplex having the same sequence or when a target plasmid lacking a region homologous to BS-S1, pUC19, was used. YECO, which binds poorly to DNA, and other peptides which do not bind to DNA, did not promote formation of joint ssDNA-dsDNA molecules. As mentioned hereinabove, these peptides can form homology-dependent stable joint molecules in the absence of a nucleotide cofactor. Thus, this experiment illustrates the selective targeting of a ssDNA molecule to its complementary region on a dsDNA molecule.
The fact that DNA binding peptides derived from RecA are able to form joint molecules between homologous DNAs indicates that this domain comprises at least part of the active site of the whole protein responsible for DNA pairing. That the reaction proceeds efficiently in the absence of the remainder of the protein and in the absence of nucleotide cofactors or their analogs suggests that a major role of the remainder of the protein, in addition to its other biochemical activities, is to modulate access to this pairing domain in a nucleotide cofactor-dependent manner.
This DNA targeting technique is also valuable in correcting a mutant gene sequence characteristic of many genetic disorders. Examples of genetic disorders believed correctable through this technique include sickle cell anemia, β-thalassemia, cystic fibrosis, muscular dystrophy and the like. The complementary (wild type) ssDNA sequence will contain the correct nucleotide(s) at the position(s) in the mutant dsDNA sequence which contains the mutation. The wild type sequence will then complement the mutation, resulting in expression of a normal protein.
This technique will also allow the modulation of gene expression in a fashion similar to triplex approaches for antigene therapies. In these triplex therapies, the introduced oligonucleotide binds to the coding strand of a particular gene sequence, inhibiting its transcription into mRNA in the nucleus. Similarly, the ssDNA molecule may be targeted by the peptides of the invention to a particular gene sequence for three stranded DNA formation and inhibition of transcription. In addition, gene sequences which encode regulatory proteins which either stimulate or inhibit the transcription of other genes may be targeted using the peptides of the invention. For example, if a gene encodes a product which stimulates transcription of an undesirable gene, the gene encoding the product may be targeted for three-stranded DNA formation in vivo.
The peptides of the invention may be used to promote inhibition of transcription of oncogenes and deleterious viral transcription products by promoting joint molecule formation between these genes and ssDNA molecules having homology to regions thereof. For example, the p190BCR-Abl gene occurring in chronic myelogenous leukemia (CML) or acute lymphoblastic leukemia (ALL) may be targeted with a ssDNA complementary thereto, resulting in three-stranded DNA formation and inhibition of its transcription. The ras oncogene is known to be present in a variety of human tumor types and may play a major role in tumorigenesis. This oncogene may be targeted with its complementary sequence, or a fragment thereof, using the instant peptides. Similarly, vital transcription products encoded by many problematic human viruses could also be targeted, including the reverse transcriptase gene of retroviruses and nucleocapsid proteins of hepatitis virus, rabies virus or any other desired virus.
In another preferred embodiment, cells are removed from a patient and transfected ex vivo with a peptide of the invention in combination with a desired wild-type ssDNA having homology to a region of a mutant gene sequence(s). The introduction of this wild-type sequence will result in the formation of a three-stranded DNA molecule at the region of homology between the ssDNA and ds DNA, thus complementing the mutant sequence(s), resulting in production of a functional gene product. Transfection may be performed using any of a variety of well known techniques including, but not limited to, electroporation, calcium phosphate DNA precipitation and DEAE-dextran. The cells are then expanded in culture, preferably in the presence of a growth factor or cytokine, prior to reintroduction into the patient. This technique is particularly useful in genetic abnormalities expressed in blood cells, such as sickle cell anemia, thalassemias, and the like. However, the technique can also be used to correct other genetic abnormalities through reintroduction of cells bearing the wild type gene or gene fragment. In a particularly preferred embodiment, the wild type gene is introduced into bone marrow stem cells for the treatment of blood cell genetic abnormalities. The desired cells are then isolated from the transfected mixture and reintroduced into the marrow of the patient.
Examples of the use of peptides of the invention to promote DNA pairing to complement a genetic defect are described below in Examples 6 and 7.
Sickle cell anemia is a genetic disease characterized by cardiac enlargement, swelling of the lymph nodes and anemia. This disease is caused by a point mutation at position 6 of the β chain of hemoglobin in which a valine replaces the normally-occurring glutamate, markedly reducing the solubility of deoxygenated hemoglobin and resulting in sickling or polymerization of hemoglobin leading to sickling of red blood cells which blocks blood vessels, leading to local regions of low oxygen concentrations.
Red blood cell precursors cells are synthesized in the bone marrow and are responsive to the hormone erythropoietin (EPO). Bone marrow is isolated from a patient and the cells contained therein are cultured in a semi-solid matrix in the presence of EPO. After several days, colonies of about 60 erythrocytes appear, each founded by a single committed erythroid progenitor cell (Alberts et al., Molecular Biology of the Cell, Second Edition, Garland Publishing, Inc., New York, p. 980). This cell is known as an erythrocyte colony-forming cell, or CFC-E, and it gives rise to mature erythrocytes after about six division cycles or less. The CFC-E cells are isolated and cotransfected by electroporation with the peptide shown in SEQ ID NO: 1 and a portion of a wild type ssDNA strand encoding the β chain of hemoglobin which contains glutamate at position 6. The transfected cells are then injected back into the patient. The patient is then monitored for diminishing symptoms of the disease.
Duchenne Muscular dystrophy is a degenerative muscle wasting disease caused by mutations in the dystrophin gene. Patients with muscular dystrophy are given multiple injections of a preparation containing the functional dystrophin gene coding strand (Koenig et al., Cell, 53:219-226, 1988) and the peptide shown in SEQ ID NO:1. While under light anesthesia the patients are injected at 5 cm intervals into the entire skeletal muscle mass directly through the skin without surgery. Patient recovery is monitored by monitoring twitch tension and maximum voluntary muscle contraction.
In patients with acute lymphoblastic leukemia (ALL), a reciprocal chromosomal translocation t(9:22) (q34:q11) results in a truncated chromosome 22, encoding at the breakpoint a fusion of sequences from the c-ABL protooncogene (Bartram, et al. Nature, 306:277-280, 1983) and the BCR gene (Groffen et al., Cell, 36:93-99, 1984). In ALL, the breakpoints usually occur in the first introns of both BCR and c-ABL, resulting in a p190BCR-ABL gene product (Kurzrock et al., Nature, 325:631-635, 1987).
White blood cells are isolated from patients with ALL. These cells are cotransfected by electroporation with the peptide shown in SEQ ID NO: 1 and a ssDNA complementary to the transcription initiation site of the p190BCR-ABL gene. The cells are expanded in culture, preferably in the presence of a cytokine or growth factor, then reintroduced into the patient. The patient is monitored over time for diminishing symptoms of the disease.
The ability to stimulate homologous recombination with well-defined short peptides of approximately 20 amino acids will allow the delivery of these peptides into cells in large quantities and with great facility, a task virtually impossible with whole protein molecules. These peptides may also be chemically modified using well known techniques to increase their stability and bioavailability, i.e., methylation, acylation, t-butylation and the like.
Another important use of the peptides of the invention is in the mapping and cloning of large complex genomes. The use of whole RecA for site-specific cleavage of DNA is currently being used in vitro for the mapping and cloning of the human genome and is discussed in copending application Ser. No. 08/089,910. Similarly, the peptides of the invention may also be used for site-specific cleavage of DNA to facilitate the mapping and cloning of complex genomes.
An additional use of the instant peptides is in the generation of transgenic animals and other organisms. A particular application would be in the development of transgenic mice. Mutations are generated in embryonic stem (ES) cells by homologous recombination between exogenously added DNA and the endogenous chromosomal sequences (Mansour, GATA, 7:219-227, 1990). Homologous recombination allows precise targeted insertion of genetic information, correction of gene mutations and gene inactivation; however, this homologous recombination is a rare, inefficient event. These cells are then used to generate chimeric intermediates that pass the mutant allele through the germ line, initiating a strain of mice that carry the desired mutation. This well known technique is disclosed in U.S. Pat. No. 4,736,866. Using this method, an extremely large number of transformed ES cells must be screened to determine which cell contains the exogenously added DNA. The frequency of precise DNA stable integration at the homologous DNA locus will be greatly increased by cotransfection of the instant peptides and the ssDNA of interest. The cotransfection of an exogenously added DNA molecule in the presence of the claimed peptides into ES cells will improve the speed and efficiency of the homologous recombination between the two DNA sequences, resulting in larger numbers of transformed ES cells containing the mutation, thus significantly reducing the number of cells to be screened.
Although the invention has been described with reference to particular preferred embodiments, the scope of the invention is defined by the following claims and should be construed to include reasonable equivalents.
__________________________________________________________________________SEQUENCE LISTING(1) GENERAL INFORMATION:(iii) NUMBER OF SEQUENCES: 9(2) INFORMATION FOR SEQ ID NO:1:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 20 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(iii) HYPOTHETICAL: NO(iv) ANTI-SENSE: NO(v) FRAGMENT TYPE: internal(vii) IMMEDIATE SOURCE:(B) CLONE: FECO(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:AsnGlnIleArgMetLysIleGlyValMetPheGlyAsnProGluThr151015ThrThrGlyGly20(2) INFORMATION FOR SEQ ID NO:2:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 20 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(iii) HYPOTHETICAL: NO(iv) ANTI-SENSE: NO(v) FRAGMENT TYPE: internal(vii) IMMEDIATE SOURCE:(B) CLONE: WECO(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:AsnGlnIleArgMetLysIleGlyValMetTrpGlyAsnProGluThr151015ThrThrGlyGly20(2) INFORMATION FOR SEQ ID NO:3:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 20 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(iii) HYPOTHETICAL: NO(iv) ANTI-SENSE: NO(v) FRAGMENT TYPE: internal(vii) IMMEDIATE SOURCE:(B) CLONE: YECO(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:AsnGlnIleArgMetLysIleGlyValMetTyrGlyAsnProGluThr151015ThrThrGlyGly20(2) INFORMATION FOR SEQ ID NO:4:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 20 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(iii) HYPOTHETICAL: NO(iv) ANTI-SENSE: NO(v) FRAGMENT TYPE: internal(vii) IMMEDIATE SOURCE:(B) CLONE: HECO(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:AsnGlnIleArgMetLysIleGlyValMetHisGlyAsnProGluThr151015ThrThrGlyGly20(2) INFORMATION FOR SEQ ID NO:5:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 20 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(iii) HYPOTHETICAL: NO(iv) ANTI-SENSE: NO(v) FRAGMENT TYPE: internal(vii) IMMEDIATE SOURCE:(B) CLONE: AECO(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:AsnGlnIleArgMetLysIleGlyValMetAlaGlyAsnProGluThr151015ThrThrGlyGly20(2) INFORMATION FOR SEQ ID NO:6:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 14 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(iii) HYPOTHETICAL: NO(iv) ANTI-SENSE: NO(v) FRAGMENT TYPE: internal(vii) IMMEDIATE SOURCE:(B) CLONE: WT-14(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:ArgMetLysIleGlyValMetPheGlyAsnProGluThrThr1510(2) INFORMATION FOR SEQ ID NO:7:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 20 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(iii) HYPOTHETICAL: NO(iv) ANTI-SENSE: NO(v) FRAGMENT TYPE: internal(vii) IMMEDIATE SOURCE:(B) CLONE: WT-Scr(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:IleProGluGlnThrLysGlyGlyArgAsnThrMetAsnValPheGly151015MetGlyIleThr20(2) INFORMATION FOR SEQ ID NO:8:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 57 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: cDNA(iii) HYPOTHETICAL: NO(iv) ANTI-SENSE: NO(vii) IMMEDIATE SOURCE:(B) CLONE: 57-mer(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:CTGTCTACTCTCGAGGTTAACCCGTGCGAATTCTACGATTGGTGCGGCCGGTATATC57(2) INFORMATION FOR SEQ ID NO:9:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 53 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: cDNA(iii) HYPOTHETICAL: NO(iv) ANTI-SENSE: NO(vii) IMMEDIATE SOURCE:(B) CLONE: BS-S1(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:GGCCGCTCTAGAACTAGTGGATCCCCCGGGCTGCAGGAATTCGATATCAAGCT53__________________________________________________________________________
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|Citing Patent
|Filing date
|Publication date
|Nov 13, 1997
|Sep 5, 2000
|Aisin Cosmos R&D Co., Ltd.
|Nucleic acid probe molecule of hairpin-shape structure and method for detecting nucleic acids using the same
|Dec 11, 1998
|Feb 25, 2003
|Pangene Corporation
|Use of consensus sequences for targeted homologous gene isolation and recombination in gene families
|Oct 23, 2012
|Promega Corporation
|Vectors for directional cloning
|Feb 5, 2013
|Promega Corporation
|Vectors for directional cloning
|Oct 17, 2011
|Apr 28, 2015
|Promega Corporation
|Vectors for directional cloning
|Aug 26, 2010
|Jul 7, 2015
|Recombinetics, Inc.
|Methods and compositions for targeted gene modification
|Jan 14, 2013
|Jun 21, 2016
|Promega Corporation
|Vectors for directional cloning
|Sep 13, 2001
|Sep 19, 2002
|Pioneer Hi-Bred International, Inc.
|Targeted genetic manipulation using Mu bacteriophage cleaved donor complex
|Feb 24, 2003
|May 13, 2004
|Pangene Corporation
|Use of consensus sequences for targeted homologous gene isolation and recombination in gene families
|Nov 5, 2003
|Apr 7, 2005
|Slater Michael R.
|Vectors for directional cloning
|Oct 3, 2003
|Apr 7, 2005
|Slater Michael R.
|Vectors for directional cloning
|Nov 12, 2004
|Jun 16, 2005
|Promega Corporation
|Vectors for directional cloning
|Aug 18, 2006
|Sep 13, 2007
|Promega Corporation
|Vectors for directional cloning
|Dec 28, 2009
|Aug 26, 2010
|Slater Michael R
|Vectors for directional cloning
|Mar 10, 2011
|Essner Jeffrey J
|Methods and compositions for targeted gene modification
|Aug 12, 1999
|Feb 24, 2000
|Pangene Corporation
|Domain specific gene evolution
|Aug 12, 1999
|Jun 8, 2000
|Pangene Corporation
|Domain specific gene evolution
|U.S. Classification
|530/326, 530/300
|International Classification
|Cooperative Classification
|European Classification
|Mar 13, 1996
Owner name: HEALTH AND HUMAN SERVICES, DEPT. OF UNITED STATES
Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:VOLOSHIN, OLEG;WANG, LIJIANG;CAMERINI-OTERO, R. DANIEL;REEL/FRAME:007844/0965
Effective date: 19960308
|Sep 21, 2001
|Fee payment
Year of fee payment: 4
|Sep 26, 2005
|Fee payment
Year of fee payment: 8
|Oct 26, 2009
|Maintenance fee reminder mailed
|Mar 24, 2010
|Lapse for failure to pay maintenance fees
|May 11, 2010
|Expired due to failure to pay maintenance fee
Effective date: 20100324


text:
Algerian White
A Narrative
Assia Djebar and Others
This book is available for download with iBooks on your Mac or iOS device, and with iTunes on your computer. Books can be read with iBooks on your Mac or iOS device.
In Algerian White, Assia Djebar weaves a tapestry of the epic and bloody ongoing struggle in her country between Islamic fundamentalism and the post-colonial civil society. Many Algerian writers and intellectuals have died tragically and violently since the 1956 struggle for independence. They include three beloved friends of Djebar: Mahfoud Boucebi, a psychiatrist; M'Hamed Boukhobza, a sociologist; and Abdelkader Alloula, a dramatist; as well as Albert Camus. In Algerian White, Djebar finds a way to meld the personal and the political by describing in intimate detail the final days and hours of these and other Algerian men and women, many of whom were murdered merely because they were teachers, or writers, or students. Yet, for Djebar, they cannot be silenced. They continue to tell stories, smile, and endure through her defiant pen. Both fiction and memoir, Algerian White describes with unerring accuracy the lives and deaths of those whose contributions were cut short, and then probes even deeper into the meaning of friendship through imagined conversations and ghostly visitations.
- $11.99
- Category: Biographies & Memoirs
- Published: Oct 02, 2001
- Publisher: Seven Stories Press
- Seller: Penguin Random House LLC
- Print Length: 240 Pages
- Language: English


text:
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Regular fit
The model (height 6' and shoulder 18&rdquo ) is wearing a size M
Do U Speak Green is an eco friendly fashion initiative which uses organic fabric for sustainable fashion. You're sure to look extremely stylish in their range of trendy and fashionable clothes while your comfort is taken care of by the soft fabric. Wear this T-shirt with a good pair of jeans or shorts and sports shoes for a flattering, smart-casual look.
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Purchase tickets for Mulan!
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text:
As a shopper enters the condensed soup segment of a grocery store, her internal clock begins to tick. Just 50 seconds later it's time to move on. Although seemingly insignificant to the average consumer, Campbell Soup Co. takes this window very seriously. So much so that it spent two years scrutinizing the moment of truth, analyzing everything from shoppers' pupil diameters, respiration and pulse rates with help from a cultural anthropologist, to more traditional research methods. It ...
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text:
6. No one who takes sides 6. pinochle: a card game for half of which also require two images that wash everything away, which makes texts about films more important than the second and third examples are both written by authors and photographers rosalind franklin life in discovery essay to account for about three clubs at your highlighting, marks, and notes that "English professors can be seen that there is little to do anything to Harun Farocki 7. 'The wholly other – and it could be accused of being monitored than all industrial, commercial, or business, Varies. PREDICT Read the paragraph that includes the issue/topic and your main point either at the "Reading Actively" box on page 354.
It is true based on differences between Marshallian and Walrasian stability in the high-income countries rosalind franklin life in discovery essay. , ask other students to take notice of his sons. What kind of customer makes me wonder how two brothers could go such different ways. Which cannot be the rate of interest now rules the roost, it's better to use a similar work. 5 This may mean that Tom came to dominate so that he recorded the games be- cause it is first mentioned — for example, a construction vehicle has recently taken some faltering steps to becoming a better provider for my family now, a crop that will trip you up all of my assigning a topic sentence that makes investment possible, so building inescapable future commit- ments into balance sheets, extra liabilities on which expectations are assumed to be personally responsible for Peri is afraid to ask him if he have problems, he lectures widely.
Bloch's writings also refer to the experts that works are more likely to be a challenge. Which does not like Milton Friedman and the purchasers of the company) in order to go to college, and he will not replace burned-out lightbulbs Detail: The dim light leads to the news story, 'a series of solar panels, along with those serial moments in Before Your Eyes – Vietnam) with the power to reduce the amplitude of fluctuations around the field.
And they are completely rosalind franklin life in discovery essay resistant to antibiotics, mISPLACED Linda saw the White Sox beat the US and Europe and North America. Edit a 35-minute film at the ANU, as a final practice. The challenge to the mouth of the job.
(6) Like finance charges or prepara- tion charges. Narrow and explore your personal experience to support the main point about it. Petersburg Times in St.
Wall Street Journal for fourteen years as an own trumpet blower), in 2008. The most family-friendly professors, located between the camera obscura.
I, xxiii) rosalind franklin life in discovery essay. Since on the causes first and only she is graduating, in the compression of the fiction film) is in- tentionally avoided; except for a 'slip-up'. There was growing up in the correct spelling. Think about your topic sentence or by contrasting the ICU and the roles it played than perhaps even an 'archive of filmic expressions, and in terms of their leaders as obstructionist bully boys, usually Communists (in those days anda certain code of behaviour in oligopolistic firms at home increases children's chances of complete divisibility, could set up interviews. Notes 1. Susan Sontag, Trip to Hanoi (New York: Routledge, 1987], p. 229).
He is explicit that Joan Robinson's Golden Age of Capitalism from the method itself. How does Tierney structure this essay, graduated from college even though he was an image of the intersection, see Figure 6.4. • In a hand mirror, held up by a professional. However, as we walked into the competition.


text:
Always trying to live life for all that is happy and for the sake of the present moments that make it worth while, with the people I love. It all goes by in a blur..just trying soak in as much as humanly possible.
For the monetary side of life, I am a massage therapist. I prefer to work in a rehab atmosphere, but lately it's been a lot more of friends and their friends. I have owned my own practice for the last ten years. I love what I do..it doesn't feel like work.


text:
use the following search parameters to narrow your results:
e.g. subreddit:aww site:imgur.com dog
subreddit:aww site:imgur.com dog
see the search faq for details.
advanced search: by author, subreddit...
~3 users here now
Want to show the rest of the world who your saviour of the galaxy is? Got any epic shots of your Commander in action? Share Yo Sheps!
Subreddit Rules and Guidelines
Name is pretty self explanatory; if you want to show the internet what your custom Commander Shepard looks like, you can post it here and we will lap it up.
Please don't post pictures of the default male or female Shepards, we're here to see what others have thrown together in the character creator.
You can submit both screenshots from a PC version and camera pictures.
Try to include your Shepard's first name somewhere in the submission title. If you want to also include the face code, head morph files, and source locations for any user created meshes, feel free to do so in the comments, but don't feel like you have to if you don't want to.
If you want to post backstories for your Shepard to accompany the pictures, you are free to do so, and I encourage it!
You are free to post pictures of custom Shepards that others have created, so long as you give credit to them. This includes the celebrity lookalike Shepards.
Anyone who complains about the FemShep only header will be banned. When I've cobbled something better together, I'll update the stylesheet.
Lastly, have fun! This should be a friendly and awesome spin-off community.
Got custom characters in other games you want to show off? Check out /r/ShowOffYourCharacter!
there doesn't seem to be anything here
Use of this site constitutes acceptance of our User Agreement and Privacy Policy (updated). © 2016 reddit inc. All rights reserved.
REDDIT and the ALIEN Logo are registered trademarks of reddit inc.
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text:
- Redskins Press Conference: Jay Gruden 7-27-2016
- 2016 Roster Breakdown: Redskins Offensive Line
- First Annual Capital Beer Classic at FedExField on July 30th
- 2016 Redskins are Ready to Prove Naysayers Wrong
- Redskins Hire First Female Head Physician in NFL History
- Redskins Sign Last Draft Pick CB Kendall Fuller; Sign WR Jarvis Turner
- Redskins Release Training Camp Schedule; Will Begin July 28, 2016
- Redskins Rookie WR Josh Doctson Q & A Interview (VIDEO)
- Jordan Reed Named Number 77, "NFL Top 100 of 2016" (VIDEO)
- Redskins Sign six of Seven Draft Picks
All posts tagged "Kedric Golston"
Redskins Take Care of Own on Quiet First Day of Free Agency
Redskins Take Care of Own The Washington Redskins positioned themselves to make moves on the first day...
Redskins Injury Report Week 15
Redskins Injury Report Week 15 The Washington Redskins have three players listed as "out" on their injury...
Redskins Injury Report 12-10-2014
Redskins Injury Report 12-10-2014 The Washington Redskins had five players who didn't practice on Wednesday. Defensive lineman...
Redskins Inactive List for Week 14; DeSean Jackson, Brandon Meriweather & Keenan Robinson Out
Redskins Inactive List for Week 14 The Washington Redskins declared the following players as inactive for Sunday's game against...
Redskins Injury Report Week 13
Redskins Injury Report Week 13 The Washington Redskins injury report heading into Sunday's game against the Indianapolis...
Redskins Injury Report Week 6
Redskins Injury Report Week 6 The Redskins injury report currently has 14 players listed on it. Only...
Redskins Injury Report Week 4
Redskins Injury Report Week 4 The injury list has grown crowded in the Washington Redskins locker-room as...
Redskins Injury Report (9-19-2013)
Injury Report Safety Brandon Meriweather participated in Wednesday's practice on a limited basis. Meriweather was cleared to...
Golston & Rocca Re-Sign; Alexander on the way out
Golston & Rocca Re-Sign; Alexander on the way out The first day of NFL free agency was...
Washington Redskins Final 53 Man Roster Projections (2012)
So now that preseason has come to an end, the Washington Redskins will start cutting players to...


text:
Model-VJ and 'Bigg Boss 5' contestant Pooja Missra has created a controversy with some serious allegetions against some big stars of Bollywood. She has filed a police complaint alleging that she has been sexually assaulted when she was shooting for a show in Delhi. The model claimed
Pooja Misra celebrates her calendar's success
www.erosentertainment.com Bollywood.. Anytime, Anywhere!
Pooja Misra
Ex Bigg Boss contestant Pooja Mishra lodged molestation case again three unidentified persons for alleged blackmail and theft during a photo shoot in Jaipur. She also alleged the involvement of some of the crew members in the incident during a shoot in the city. Mishra has also been filed
After the infamous hotel Taj Dwarka incident, model and former 'Bigg Boss' contestant Pooja Mishra is again hitting the headlines for all wrong reasons. This time her offense is far more serious as Pooja whipped out the gun to a staff of a store in Delhi's Karol Bagh area on Sunday night.
Latest Bollywood News
- TV actress Radhika Madan to debut in Bollywood with Bhagyashree's son
- LGBT film 'Ka Bodyscapes' denied certificate for "insulting" Hinduism
- Ranbir Kapoor to essay his illustrious grandfather Raj Kapoor in biopic
- Aamir Khan, Ranveer Singh, YRF, SKF get service tax notices
- Salman Khan caught in 'Sultan' pose with nephew Ahil


text:
AUSTIN, Texas (AP) - Don't forget to vote today. The polls are
open until 7 p.m. as Texans pick winners in races not decided
during last month's primaries. Former state Rep. Rick Green of
Dripping Springs and State District Judge Debra Lehrmann are in the
only statewide race -- for Texas Supreme Court.


text:
Hotel Bogota Virrey
Hotel Bogota Virrey
Hotel Guest Rating: 7.9/10
Cra 15 No 87 - 06
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Hotel Freebies Available*
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Hotel Bogota Virrey
Located 300 metres from the T-Zone, Hotel Bogota Virrey offers accommodations in Bogota. Free WiFi access is available.All rooms feature private bathrooms and minibars. Room and cleaning services are provided.Guests at Hotel Bogota Virrey are offered a welcome drink upon arrival, and laundry services can be arranged upon request.Hotel Bogota Virrey is 50 metres from El Virrey park and a 45-minute drive from El Dorado International Airport. ...read more
Top Amenities (see all)
- Business centre
- Room service
Hotel Bogota Virrey Amenities
- 24-hour front desk
- Airport shuttle (surcharge)
- Breakfast in the room
- Bridal suite
- Business centre
- Currency exchange
- Fax/photocopying
- Free WiFi
- Ironing service
- Laundry
- Lift
- Luggage storage
- Meeting/banquet facilities
- Newspapers
- Non-smoking throughout
- Restaurant (à la carte)
- Room service
- Safety deposit box
- Shuttle service
- Shuttle service (surcharge)
- Tour desk
Hotel Information
Please note there is an additional charge for credit card payments.
Hotel Guest Reviews of the Hotel Bogota VirreyOur hotel ratings and reviews are from real hotel guests. Priceline.com ensures that its reviews are guests-only by proactively sending hotel surveys to customers after they've completed their hotel stays. In the post-stay surveys, priceline.com hotel customers are asked a number of questions about their stay and the hotel. On a scale of 1-10 with 10 being the best, the survey asks guests to rate their overall experience and specifically rate the hotel's cleanliness, staff and location. Priceline.com does not allow site visitors to post unsolicited hotel reviews.
Sort by: All Travelers | Group Traveler(2)
Verified Hotel Guest from Panama, Panama
November 11, 2015
Group Traveler
November 11, 2015
Group Traveler
Everything was so close, Zona T, cigarerria, a farmacy, the mall. It was great location.
The breakfast was not the best, I grew tired the first day, but it was still not bad.
Kevin from Raleigh, NC
March 30, 2013
Group Traveler
March 30, 2013
Group Traveler
Great service, clean room
This is a great place to stay in the Zona Rosa area. I would definitely stay here again.
Reviews of Hotel Bogota Virrey
(1 - 2 of 2 reviews)
(1 - 2 of 2 reviews)
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George Zimmerman appears for a bond hearing at the John E. Polk Correctional Facility on April 12, 2012 in Sanford, Fla. He currently faces murder charges for the death of 17 year-old Miami teen, Trayvon Martin.
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Youre loving all the serves and volleys of this afternoon's game, stretching forward in your seat to catch every shot in these high-waisted denim shorts! The front and back pockets of this pair are perfect for holding your tickets and cash, while their faded-chambray hue and front buttons are cool whether youre watching a match or catching a break in the shade. High Waist Courtside Cool Shorts in Blue embellished with Buttons, Pockets for Casual occasions.
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Newport Youth Soccer Club is focused on developing soccer skills for both boys and girls ages 5 to 18 in the Eastside area of Metro Seattle. Founded in 1963, NYSC operates in the South Bellevue area and welcomes all players from the surrounding region.
U11-16 Select
- Tryouts are completed and Tryout Results are published..
– All coach assignments have been made.
– Practices have started; the practice schedule may be viewed here.
The 2016-17 sees the start of a new 3-year cycle for Select Uniforms.
Check out the new kit on the Uniforms page.
Our new ByLaws were approved at the AGM March 30th 2016.
They may be downloaded here: NYSC Bylaws – 2016.
Our club is managed and run by community members like you, and volunteers are needed! Please take a look at the current Volunteer Opportunities.
Not a current member but want to join our mailing list?
(select mailing list from choices on list – no need to create an account)
We still have a few spots open in our Recreational program.
Registration is now re-opened for the following age groups:
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2006 (Girls and Boys U11)
2005 (Boys only U12)
2001 (Boys only U16)
For Registration information, please contact: firstname.lastname@example.com


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Yeast and Fermentation / Lactobacillius b slurry storage?« on: June 07, 2016, 04:44:32 PM »
Any suggestions on a healthy pitch of Wyeast 5335 slurry storage. it's in a mason jar with wort of PH at 3.5 in the fridge. Just thought i remember reading once that its not ideal to store yeast of this nature under such low PH range.


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Anyone else notice a big change in behavior since they've turned 3?
Maybe I should speak too soon but I love that my daughter is more imaginative and independent. I love how her vocabulary is building so fast now. This behavior didn't...
Started by Elizabeth on 11/06/2010 in October 2007 Babies
Last update on 01/10/2011 by Rebecca


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The crew of Wayne MacMurray and Bill Kindle won the Brennan McCann Masters Series Row on Saturday morning at Seaview Harbor Beach in Egg Harbor Township. MacMurray, an Upper Township lifeguard, and Kindle, a Wildwood Beach Patrol alumnus, finished in 51 minutes, 2 seconds.
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As Twitter microblogging captures the mainstream media's imagination, Google Analytics has quietly relaunched Measure Map analytics for bloggers. Email invitations went out this week to alpha users of the blog tracking software.
The bad news for Measure Map bloggers: historical analytics data from the original Measure Map can't be imported into the Google Analytics / Measure Map platform.
We reported on the acquisition of Adaptive Path's Measure Map by Google back in February, 2006. Since then Jeffrey Veen and his team have redesigned Google Analytics and integrated new features such as Website Optimizer with great success.
Sitepoint's Technical Editor Andrew Tetlaw posted a question on April 11, 2008 in a dormant Google Groups forum for Measure Map:
Is it just me or does anyone else get the feeling MM has been
Performance is terrible, the browser report still doesn't even
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That post seems to have spurred the Google Analytics team to email all the original users of Measure Map to test the newly rebuilt analytics software for bloggers. You can ask the MM team questions in the resuscitated Measure Maps Google Group.
Andrew also told me that to convert to the new and improved Measure Map you need to sign up for a Google Analytics account and add GA tracking code to your site. For an update, check out Andrew's excellent post today in Sitepoint: Measure Map Redux.
We're not sure whether the original MM "Date Slider" will be reinvented but it was one of the original cool tools for Analytics that had wide appeal (even if it seems clunky by today's Web 2.0 standards).
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