HIV‐1 integrase specifically recognizes and cleaves viral end DNA during the initial step of retroviral integration. The protein and DNA determinants of the specificity of viral end DNA binding have not been clearly identified. We have used mutational analysis of the viral end LTR sequence, in vitro selection of optimal viral end sequences, and specific photocrosslinking to identify regions of integrase that interact with specific bases in the LTR termini. The results highlight the involvement of the disordered loop of the integrase core domain, specifically residues Q148 and Y143, in binding to the terminal portion of the viral DNA ends. Additionally, we have identified positions upstream in the LTR termini which interact with the C‐terminal domain of integrase, providing evidence for the role of that domain in stabilization of viral DNA binding. Finally, we have located a region centered 12 bases from the viral DNA terminus which appears essential for viral end DNA binding in the presence of magnesium, but not in the presence of manganese, suggesting a differential effect of divalent cations on sequence‐specific binding. These results help to define important regions of contact between integrase and viral DNA, and assist in the formulation of a molecular model of this vital interaction.
Integration of a DNA copy of the human immunodeficiency virus (HIV) genome into the host genome is a vital step in HIV replication. After reverse transcription of the viral genomic RNA, two nucleotides are cleaved from each 3′ end of the viral DNA adjacent to a conserved CA dinucleotide. Following this 3′ processing reaction, the exposed 3′ hydroxyls are used in a direct transesterification reaction called strand transfer, in which the 3′ viral ends are joined to 5′ phosphates in the host DNA to produce the integration intermediate. After integration, the remaining unpaired nucleotides at the 5′ ends of the viral DNA are removed, and the single‐stranded region of DNA between the integrated virus and the host DNA is repaired; both of these steps are presumably accomplished by host enzymes. For recent reviews on retroviral integration, see Katz and Skalka (1994), and Vink and Plasterk (1993).
Human immunodeficiency virus type 1 (HIV‐1) integrase can catalyze both the 3′ processing and strand‐transfer reactions in vitro using oligonucleotide substrates which mimic the viral DNA ends (Sherman and Fyfe, 1990; Bushman and Craigie, 1991; LaFemina et al., 1991). Integrase can also carry out an apparent reversal of strand transfer which has been termed disintegration, in which a branched substrate which mimics the product of strand transfer can be resolved into viral and target DNA segments (Chow et al., 1992). Mutagenesis and proteolysis experiments have shown that integrase consists of three functional domains (Engelman et al., 1993); structural information is now available for all three pieces of the protein. The N‐terminal domain contains a conserved HHCC motif which binds zinc with a stoichiometry of 1:1. Binding of zinc to the N‐terminal domain enhances the multimerization of the full‐length protein and increases the activity of the protein (Burke et al., 1992; Haugan et al., 1995; Zheng et al., 1996). The structure of a dimer of the N‐terminal domain was solved by heteronuclear NMR (Cai et al., 1997). Each monomer consists of a helix–turn–helix motif, in which the helix normally used by this class of proteins to recognize specific DNA sequences is instead used to form protein–protein contacts that create the dimer interface. The catalytic core domain contains three acidic residues, D64, D116 and E152 which are essential for catalytic activity (Engelman and Craigie, 1992; Kulkosky et al., 1992). These residues constitute a D,D‐35,E motif which is conserved among all retroviral integrases, retrotransposons and some bacterial transposases (Rowland and Dyke, 1990; Polard and Chandler, 1995). The crystal structure of the HIV‐1 core domain has been determined by X‐ray crystallography, and has been shown to be a member of a superfamily of polynucleotidyl transferases, including Mu transposase, Escherichia coli RuvC, and E.coli RNase H, which are proposed to use these three catalytic acidic residues to coordinate metal ions during catalysis (Dyda et al., 1994; Yang and Steitz, 1995; Bujacz et al., 1996; Rice et al., 1996). The C‐terminal domain, which displays a much lower level of sequence homology among retroviruses, has been shown to bind DNA in a non‐specific fashion (Vink et al., 1993; Woerner and Marcus‐Sekura, 1993; Engelman et al., 1994). The NMR structure of this domain reveals a dimer with an SH3‐like fold, which produces a large cleft that has been proposed to bind DNA (Eijkelenboom et al., 1995; Lodi et al., 1995).
In order to catalyze cleavage and strand‐transfer reactions, integrase must first distinguish between substrate and target DNA; that is, between specific viral end DNA, and non‐specific host DNA. Stable complexes of integrase and viral DNA ends have been observed in the presence of divalent metal ions, and require the intact integrase protein (Ellison and Brown, 1994). These complexes are stable in the presence of competitor DNA, can be formed on either the U3 or U5 DNA substrates, and require <15 bp of DNA at the viral termini (Ellison and Brown, 1994; Vink et al., 1994). Numerous studies have been carried out in which viral DNA end sequences were altered, and effects on processing and integration were monitored, but the results of these studies are contradictory. Several investigations have suggested that there is limited sequence specificity required, with only the well conserved CA dinucleotide being needed for processing and strand transfer (LaFemina et al., 1991). Other studies have suggested the importance of 2–7 base pairs upstream of the conserved CA, with varying effects on the reaction (Vink et al., 1991; van den Ent et al., 1994; Yoshinaga and Fujiwara, 1995; Katzman and Sudol, 1996a; Balakrishnan and Jonsson, 1997). In vivo studies have not significantly clarified these results, although the majority of them seem to indicate that more than just the conserved CA residue are needed (Murphy et al., 1993; Reicin et al., 1995). There are several possible reasons for the contradictory results. First, many of these experiments failed to examine all possible base substitutions in the LTR region, which could lead to erroneous assumptions about specificity at certain positions. Secondly, all of the in vitro studies were carried out with assays using manganese as the divalent metal cofactor. More recently, assay conditions utilizing magnesium as a cofactor have been developed (Engelman and Craigie, 1995). Since magnesium is likely to be the relevant divalent cation used by integrase in the cell, this assay should provide a more realistic way to measure effects on activity. In addition, a number of related enzymes are known to have dramatically altered DNA‐binding specificity or activity in the presence of manganese compared with magnesium (Hiom and Gellert, 1997; Junop and Haniford, 1997).
In order to better understand the basis for viral end DNA‐binding specificity, we have examined all of the possible viral DNA end mutations for their effect on integrase activity in the presence of either magnesium or manganese. In addition, we have developed a system in which integrase itself can be used to select for sequences that it prefers under different conditions. In a previous study, we used specific photocrosslinking to identify a vital contact between the conserved adenosine residue in the viral DNA end and K159 of integrase (Jenkins et al., 1997). We now extend those studies to examine potential regions of interaction along the viral end DNA. These three methods taken together provide a much clearer picture of how integrase binds the viral end DNA, and should help us to better model the critical interactions of amino acids on the protein with specific regions of the viral DNA.
The rate of 3′ processing activity of HIV‐1 integrase was determined using two separate sets of reaction conditions, and two different metal cofactors. The maximal rate of processing (1.22 ± 0.18 fmol/min) was observed in reactions using the magnesium buffer conditions in the presence of magnesium (Figure 1). With the same buffer, activity was also observed with manganese as cofactor; however, the rate of processing was reduced 2‐fold (data not shown). Processing in the manganese buffer conditions with manganese as cofactor (0.84 ± 0.11 fmol/min) was ∼70% of that seen in magnesium buffer conditions. This same ratio of processing activity between the magnesium and manganese supporting buffers was seen at several different concentrations of integrase, and both reactions showed a similar optimal rate of processing near 1 μM integrase. As expected, no activity was detectable in either buffer in the absence of any metal ion. The extent of processing in both the magnesium and manganese assays was linear over a period of 60 min, and in both cases, all of the substrate could be completely converted to cleaved product.
Comparison of 3′ processing rates in Mg or Mn buffers. Time‐courses of integrase activity were performed as described in Materials and methods. Aliquots were removed at the indicated times and the amount of product produced was determined by autoradiography. The vertical axis is presented as % product formed, which was calculated as the ratio of radioactivity in the product band to the sum of the radioactivity in the product and substrate bands. The indicated data points are the average of three separate determinations; error bars indicate the standard deviation. Calculated reaction rates were determined by a least squares fit of the first seven timepoints (out to 60 min); in both cases, the r2 value for the fit is >0.98.
In order to identify elements of sequence specificity for the 3′ processing reaction, mutations were introduced into the U5 LTR sequence. All possible single mutations were made in the terminal 16 bases of the LTR (Figure 2A); in all cases, double‐stranded substrates contained the proper complementary base pairs at the site of mutation. For each of the 64 mutant substrates, integrase reactions were carried out and initial rates of 3′ processing were determined. Reactions were carried out at least three times per substrate, and an average rate was calculated. This rate was compared with the rate of reaction of the wildtype U5 LTR substrate to determine a percentage of wildtype value for each mutant. This process was carried out in both the magnesium and manganese assays. The results of this analysis are presented in Figure 2B, C, D and E.
Relative 3′ processing rates of mutated HIV‐1 LTR sequences. (A) Sequence of the top strand of the wildtype HIV U5 and U3 LTRs, with the numbering of the positions of the terminal 15 bases. The position of the 3′ processing cleavage is shown by the arrow. (B–E) Effects of mutations of the various positions of the LTR on 3′ processing rates. The number and letters along the horizontal axis signify the position and type of mutation. The vertical axis expresses the initial rate of processing of the mutant as a percentage of the wildtype initial rate (using the U5 LTR as the wildtype substrate). Initial rates were generally calculated using timepoints out to 60 min; in the few cases where r2 values for the 60 min points were <0.95, only timepoints out to 45 min were used. One hundred percent signifies that the mutated substrate is processed with the same rate as the wildtype U5 substrate. Each bar corresponds to the average of three separate rate determinations on a given mutant LTR sequence. Black bars represent reactions carried out under Mg conditions, while gray bars indicate reactions carried out under Mn conditions. Percentage of wildtype rates were based on wildtype reactions using the same metal ion as the mutated substrates. Error bars show the standard deviation of the three measurements.
Changes in sequence at several positions showed no effect on 3′ processing. These included positions 12, 13 and 14, for which the activity observed in the mutants was similar to that of the wildtype U5 substrate (data not shown). Additionally, the mutants at positions 5, 6 and 7 were cleaved at rates between 90 and 110% of the wildtype levels, suggesting that these sequences had no significant effect on the processing reaction (Figure 2C and D). These results were observed in both magnesium and manganese assays. Figure 2B shows that mutations at positions 10 and 11 had no significant effect in the manganese assay, but dramatically affected 3′ processing in the magnesium assay. In particular, substitutions of guanine at position 11 reduced rates of processing to <10% of the wildtype substrate, while all three substitutions at position 10 reduced levels of processing by at least 2‐fold. Position 9 showed an intermediate effect, in which neither assay was dramatically affected by changes to adenine or guanine, whereas a mutation to cytosine reduced processing levels 2‐fold in the manganese assay, and nearly 20‐fold in the magnesium assay. Position 8 (Figure 2C) showed a similar effect, with one mutation (cytosine to guanine) producing a significant change in processing rate.
Nucleotides closer to the 3′ end of the substrate were also adversely affected by mutations. Specific mutations in positions 3 and 4 (guanine and thymine, respectively) reduced activity by 5‐ to 15‐fold (Figure 2D). Here, a differential effect of magnesium and manganese on the changes in activity was not observed. Position 2 (Figure 2E), a cytosine which is conserved throughout all retroviral LTR sequences, produced the anticipated results; all three mutants were poor substrates for processing, with almost no activity observed in the magnesium assay, and <10% of wildtype activity in manganese. The conserved adenine at position 1 was completely essential under both assay conditions; none of the mutant substrates was able to be processed (data not shown). The −1 and −2 positions also had strong specificity requirements (Figure 2E). Position −1 favored the presence of a guanine or thymine; cytosine and adenine were tolerated in the manganese assay, but rates of processing were reduced 10‐fold under magnesium conditions. Position −2 was only marginally affected by any mutations in the manganese assay, but under magnesium conditions, the cytosine mutant was strongly defective for processing.
An alternate means to examine substrate specificity is to allow integrase to 'choose' its own preferred substrates. To do this, we carried out a modified ligation mediated PCR (LM–PCR) reaction as described in Materials and methods, and outlined in Figure 3. Four sets of degenerate oligonucleotides were used to analyze the processing specificity. The four substrates each had three degenerate bases; substrate 1 at positions 9–11, substrate 2 at positions 6–8, substrate 3 at positions 3–5, and substrate 4 at positions 1 and 2. After creation of the double‐stranded substrates by addition of DNA polymerase, integrase was added to allow 3′ processing to occur. Processed substrates were then captured by ligating a duplex linker with a 2 bp 5′ overhang complementary to the 5′‐CA overhang of the cleaved processing product. LM–PCR then amplified the ligated fragments, ensuring that only processing products were analyzed. After subcloning, these products were sequenced to determine which sequences were utilized by integrase. As a control, a template containing a sequence which was not cleavable by integrase was used; no LM–PCR products were detected with this template.
Schematic diagram of the in vitro LTR selection scheme. Only portions of the actual DNA sequences are shown for the sake of clarity. In step 1, a short oligonucleotide is annealed to a degenerate U5 LTR bottom strand. This duplex is extended with DNA polymerase in step 2 to form the substrate, which is reacted with integrase in step 3 in the presence of either Mg or Mn to form the cleaved product with protruding 3′ hydroxyl. In step 4, a linker with a 2 bp 5′ overhang is ligated to the product; the gray triangle emphasizes that there is no covalent linkage between the 3′ hydroxyl of the adenosine and the 5′ hydroxyl of the guanosine on the linker top strand. In step 5, PCR primers are added to the reaction, and LM–PCR is carried out to produce the final product.
Between 9 and 16 individual clones were analyzed for each substrate, and the same selection procedure was carried out using both the magnesium and manganese assay conditions; a summary of the sequences found at each position is shown in Table I (Mg) or Table II (Mn). Figure 4 graphically compares the results of the selection experiment with the results of the analysis of individual LTR mutations. Regions of the LTR which have effects on processing rates are marked with circles. The intensity of the shading of the circles shows the relative significance of the effect, with open circles being a minor effect (or effects from only one nucleotide mutation), shaded circles signifying regions that are strongly reduced for processing when mutated, and black circles for bases which appear critical for processing. Because of the design of the LM–PCR experiment, the two most 3′ positions (−1 and −2) could not be tested in the in vitro selection system, as these bases must remain the same for the ligation linker to anneal properly.
Comparison of the regions of the viral LTR important for 3′ processing under Mg or Mn conditions. The sequence of the wildtype HIV U5 LTR is shown at the top. Circles represent positions which when mutated produce an effect on 3′ processing rates. Open circles represent the weakest effect, including small changes (<40%) in activity or effects seen only with specific mutations. Shaded circles represent a moderate effect; mutations in these residues produce dramatic reductions in processing rates and/or reductions are seen with multiple changes at that position. Black circles represent the strongest effects; mutations in these residues nearly eliminate processing activity. The top two lines represent results using the Mg conditions, while the bottom two lines represent the same results in the Mn conditions. The top line in each group presents a summary of the data from the complete set of LTR mutants (Figure 2), while the bottom line summarizes the results seen in the in vitro selection assay (Figure 3). Positions −1 and −2 could not be examined in the in vitro selection assay.
To analyze further the regions of both protein and DNA required for viral DNA binding by integrase, we placed specific photocrosslinking agents at various positions along the DNA substrates. Figure 5A shows the location of the 5‐iododeoxyuracil (IdU) or 5‐iododeoxycytosine (IdC) residues (boxed regions). Each substrate had only one unique position replaced by the photocrosslinker. Figure 5B shows a representative autoradiogram of a gel separating the products of a 30 min crosslinking reaction. In several cases, distinct bands corresponding to the molecular weight expected for a protein–DNA complex were seen. A control reaction, containing DNA without the photocrosslinker, produced no shifted material. The level of crosslinking (defined as the percentage of radioactivity in the product band relative to the total radioactivity in the lane) ranged from <1% [for C(2)] up to nearly 10% [for A(−2)]. The amount of crosslinking at position A(1), which has been previously described and is not shown in this figure, was approximately 10% (Jenkins et al., 1997). These crosslinking reactions were carried out under magnesium conditions; under manganese conditions, specific crosslinking was only observed with three substrates: A(−2), C(−1) and A(1), and in all three cases the level of crosslinking was <1% (data not shown).
Results of photocrosslinking studies on the HIV U5 LTR. (A) The sequence of the wildtype U5 LTR. Boxes indicate residues which have been replaced uniquely with photocrosslinking reagents in the various substrates. Thymine and adenine residues were replaced with 5‐iododeoxyuracil (IdU), while cytosines were replaced with 5‐iododeoxycytosine (IdC). (B) The autoradiogram of a 4–12% NuPage MES gel of crosslinked samples. Lanes are identified by the position of the crosslinker in the substrate DNA; for example, T(7) represents the replacement of the thymine at position 7 with an IdU residue. 'Con' is a control DNA containing no photocrosslinkable residues. 'S' represents the position of the substrate DNA, and 'X' represents the position of the protein–DNA crosslinked complex. (C) Results of the crosslinking experiments. Residues with detectable amounts of crosslinking are boxed. The four strongest crosslinking signals are marked with filled circles. The peptides or amino acids which were crosslinked at these positions, as determined by N‐terminal sequencing, are indicated. The A(1) crosslink to K159 was described in a previous work, and is not represented in the figure in (B). (D) Plot of the per cycle yield of PTH‐amino acid derivatives from N–terminal sequencing of the A(−2) crosslinked peptide. Crosslinked residues are represented by black bars. The diagonal line represents the best‐fit linear regression of the cycles excluding the two crosslinked residues. Serine residues naturally fall below the regression line due to hydrolytic loss of derivatives.
In order to identify the specific regions of integrase that were interacting to form the crosslinks, integrase–DNA complexes were isolated and proteolyzed, and labeled crosslinked peptides were N‐terminally sequenced. Figure 5C identifies the regions of the DNA which were sensitive to crosslinking, and the locations of four separate specific crosslinks. In two cases, we were able to specifically determine the residue(s) which interacted with the crosslinked position. The crosslink to K159 at position A(1) has previously been described (Jenkins et al., 1997). The strong crosslink observed at position A(−2) was mapped to a peptide containing amino acids 139–152. N‐terminal sequencing of this peptide produced the data shown in Figure 5D. There is a significant absence of the expected PTH derivative at the position corresponding to tyrosine 143. In addition, there is a decrease in the expected amount of the derivative in the cycle corresponding to Q148. Using the repetitive yield to calculate expected amounts of PTH derivatives, we estimate that 75% of the crosslinking at this position is to Y143, and the other 25% to Q148.
The crosslink to C(−1) could not be isolated to a specific amino acid. The crosslinked peptide began at H51, and based on the cleavage reagents and fragment size, likely ended after position 64. Sequencing produced data up to I60, but no sequence was observed C‐terminal of that position. This could be due to crosslinking which somehow disrupted the amino acid chain, but is more likely due to premature elution from the sequencing column. Other data suggest that peptides of less than 4 or 5 amino acids often elute from the column during washing. Therefore, we could not isolate the specific amino acid, but crosslinking was likely to be to W61, Q62 or L63. The minimal peptide that was involved in the T(5) crosslink was identified as 247–270. However, the lower amounts of crosslinked material prohibited assignment of a specific region of crosslinking, as clear Edman data were only seen through position 254. In the other three significant crosslinks, the level of crosslinking was either too low to permit accurate determination of the location, or crosslinking occurred to multiple regions of the protein (data not shown). Figure 6 graphically compares the results of the three magnesium‐based experiments. As in Figure 4, shaded circles are used to represent the relative amount of specificity requirements on a given position of the LTR. Levels of crosslinking were used as an indication of the relative importance of interactions at given positions in the photocrosslinking study.
Summary of the critical regions of the viral LTR for 3′ processing under Mg conditions. The sequence of the wildtype HIV U5 LTR is displayed at the top. Circles represent positions which when mutated produce an effect on 3′ processing rates as indicated in the legend to Figure 4. The three sets of data represent results of the mutated LTR study (Mutation), the in vitro selection (Selection) and the photocrosslinking (Crosslink). Where known, the regions of the protein which interact with the different parts of the LTR are indicated.
HIV‐1 integrase 3′ processing and strand transfer occur in vitro in the presence of either magnesium or manganese. Early reports suggested that activity was more robust in manganese (Sherman and Fyfe, 1990; van Gent et al., 1992); however, under optimal conditions, we observe activity in magnesium which is slightly higher than that seen in manganese. Several lines of evidence suggest that DNA binding, and probably sequence specificity, are differentially affected by the type of metal ion used. It has been shown that HIV‐1 integrase has a non‐specific nuclease activity when in the presence of manganese; similar activity is not observed in magnesium (Engelman and Craigie, 1995). In addition, in the presence of manganese, integrase is capable of using alternate nucleophiles to carry out the 3′ processing reaction, including alcohols such as glycerol or methanol (Katzman and Sudol, 1996b). In magnesium, water is the predominant nucleophile used by the protein. Specific photocrosslinking is also dramatically reduced in the presence of manganese as compared with magnesium (D.Esposito and R.Craigie, unpublished results). These facts suggest that the active site of the enzyme may be considerably less 'tight' in the presence of manganese, allowing a wider selection of nucleophiles, and potentially reducing the DNA‐binding specificity of the 3′ processing reaction. This would also help to explain why the disintegration activity observed in vitro, which involves the apparent reversal of the strand‐transfer reaction, occurs only in the presence of manganese and appears to require no sequence specificity beyond the CA dinucleotide (Chow et al., 1992; Balakrishnan and Jonsson, 1997). Many other DNA‐binding proteins involved in polynucleotide transfer show dramatic effects when manganese is substituted for magnesium, ranging from alterations in DNA‐sequence specificity for the eukaryotic RAG proteins (van Gent et al., 1996; Hiom and Gellert, 1997) to effects on catalytic activity or reacton kinetics for the MuA and Tn10 transposases (Savilahti et al., 1995; Junop and Haniford, 1997). One report has also shown a change in sequence specificity of RSV integrase in the presence of magnesium (Katzman et al., 1989). These arguments suggest that many of the effects of sequence changes seen in HIV‐1 integrase under the commonly studied manganese conditions may not mimic the effects that are important in the presence of magnesium, likely to be the essential cation in vivo. In order to examine this possibility, we constructed LTR substrates which had mutations in every possible position 14 bases in from the 3′ end, and analyzed their ability to be processed by integrase under magnesium or manganese conditions. The results clearly show a differential effect of the cation on specific locations along the LTR, and also very strong effects of particular nucleotide replacements on activity. As added confirmation, we used an in vitro selection procedure to reproduce very similar results under both cation conditions.
Perhaps the most interesting result of the mutational studies was the apparent importance of residues very distal to the LTR end, at positions 8–11. In the past, it has been reported that regions >6–8 bases from the end of the viral DNA were not required for integration (LaFemina et al., 1991; Vink et al., 1991; van den Ent et al., 1994; Balakrishnan and Jonsson, 1997); these reports using direct assays and methylation interference were all done under conditions in which manganese was the divalent cation. Under these conditions, we saw little effect of these mutations on activity. Only in two specific cases, T9C and C8G, was there any significant effect in the presence of manganese, and even with these mutants the reduction in activity was no more than 2‐ to 3‐fold. In contrast, with magnesium, activity was dramatically affected by mutations at positions 9, 10 and 11 (Figure 2B). It is important to note that the U3 sequence differs from the U5 sequence at positions 9 and 11. In both cases, the nucleotide present in the U3 sequence (T at position 11, G at position 9) shows the least effect on activity in these experiments, as would be expected. The data obtained from the in vitro selection experiment (Tables I and II) corroborate the mutational data. Under manganese conditions, no sequence specificity was observed at any position upstream of position 4, while in the magnesium conditions, very clear selectivity was seen from positions 8–11, with similar base preferences as predicted by the mutational study. The results of the selection experiment also help to suggest that the effects of mutations at various positions appear to be mainly isolated to those positions. Since we are mutating single bases, there is always the possibility of synergistic effects of mutations at different positions. The fact that the selection experiment produces results very similar to those seen with the single mutations suggests that there is little synergy within the LTR. Mutations at positions 12–14 (data not shown) failed to have any effect on the in vitro 3′ processing activity in the presence of either cation.
Mutations at positions 6 and 7 had no effect on 3′ processing activity (data not shown) and these positions showed no specificity in the in vitro selection in the presence of either cation. Position 5 mutations also had no effect on 3′ processing activity, and this position did not appear to favor any particular base in the in vitro selection under manganese conditions. However, there did appear to be a strong preference in the presence of magnesium for the use of cytosine or thymine at this position. Thymine is the wildtype base in both U5 and U3 LTRs, but the activity of the T5C mutant in the mutational study is actually slightly higher than the wildtype rate, and was in fact the most active of all the mutant substrates. Mutations at positions 3 and 4 did have deleterious effects in both the magnesium and manganese assays, and in most cases there was little differential effect between the two cations. The selection experiments confirmed that A was the most commonly selected base at position 4 with both cations, and G was not observed at this position. The apparent lower activity of U3 substrates in vitro may in part be explained by the presence of a thymine at position 4, which has a small but significant effect on activity in the mutational data, and is not nearly as favored in the selection. Likewise, G and C were most commonly seen at position 3, and T was not selected. These results are qualitatively similar to published results using manganese.
The 'invariance' of the CA dinucleotide has been well established in previous studies (Vink et al., 1991; van den Ent et al., 1994; Yoshinaga and Fujiwara, 1995; Balakrishnan and Jonsson, 1997), and the results of this study confirm those data. The effects of mutations on position 2 are dramatic. In the presence of magnesium, only a C2G mutation shows any activity, and that is down >20‐fold from wildtype levels. In the presence of manganese, slight activity can be detected with any of the three mutations, but processing is always significantly impaired. Position 1 mutations were not tolerated under any cation conditions; no mutation of position 1 produced any detectable activity, demonstrating the complete requirement for an adenosine at this position. It is interesting to note that replacement of the A–T base pair at position 1 with a mismatched base pair of T–T does permit activity; in magnesium the level is ∼20% of wildtype, while in manganese it is slightly higher. Previous results have demonstrated the likelihood that this base (as well as those at positions −1 and −2) is unpaired during the reaction (Scottoline et al., 1997), suggesting that the increase in activity with the mismatched bases may be a result of easier separation of the two strands.
The two terminal positions demonstrated interesting specificity requirements, with particular substitutions having very deleterious effects. However, it was impossible to confirm these results by means of the in vitro selection procedure, since the sequence of these bases was required to be fixed for proper ligation during LM–PCR. These positions are particularly interesting since they vary greatly among the different retroviruses, and may strongly impact on the sequence specificity of the reaction.
Specific photocrosslinking has been used to reveal important protein–DNA contacts in several systems, including the interaction of HIV‐1 integrase with DNA (Willis et al., 1993, 1994; Jenkins et al., 1997). We have chosen 5‐iododeoxyuracil and 5‐iododeoxycytidine for a number of reasons, including their longer wavelength of crosslinking, which limits photodamage to the protein, and their chemical and steric similarity to natural DNA bases. Most importantly, these reagents are 'zero‐distance' crosslinkers, which must be nearly within van der Waals contact to their target in order to form stable crosslinks (Deitz et al., 1987). Therefore, they avoid the complications which arise with linker‐arm crosslinking agents which can crosslink 5–10 Å away from the DNA, and should reveal more detailed regions of interaction. Placement of the crosslinkers at various positions along the U5 LTR revealed numerous regions of crosslinking, with varying amounts of crosslinked product being formed. As expected from the results of the mutational studies, many of the strongest crosslinks were observed in regions which were highly sensitive to mutation, including the terminal LTR region. The strongest crosslinks were seen to the A at position 1, but significant levels of crosslinking to the A at the −2 position and the C at −1 were also observed. A region in the proximal LTR at positions 4 and 5 also produced high levels of crosslinking. Finally, the two iodouracil residues placed at positions 10 and 11 showed lower, but clearly detectable levels of crosslinking, suggesting that this region is also involved in contacts with the DNA, as was predicted from the magnesium mutational data. Conspicuously absent were crosslinks to the C(2) position, which might be expected due to the importance of that position to specificity. There are several possible reasons for the lack of crosslinking. First, the residue(s) involved in this interaction may be poorly crosslinkable to iodocytosine. Alternatively, the interaction at this region may involve the positioning of water or metal ions, which may place the actual amino acid outside the distance of the crosslinking agent. Finally, the interaction may take place to a region away from the 5‐position of the cytosine, and again, may be outside the optimal crosslinking distance. It is possible that other crosslinking agents may be more successful at identifying interactions at this position. Interestingly, little crosslinking was observed in the presence of manganese at any position other than positions 1, −1 and −2, and these positions produced crosslinking at levels 3‐ to 5‐fold lower than in magnesium. This suggests that in the presence of manganese, the DNA is in fact less tightly bound to the protein, or that the complex of integrase with DNA is more transient, and thus less likely to crosslink.
Samples which produced significant levels of crosslinked product were analyzed by proteolysis and N‐terminal sequencing to identify regions of contact. The results help to clarify some of the mutational data, and identify critical protein–DNA interactions. The terminal bases all seem to be involved in contacts with the catalytic core of the protein, while the only analyzed proximal crosslink is clearly contacting the C‐terminal portion of the protein. These data are consistent with crosslinking results seen by Heuer and Brown using disintegration‐type substrates involving target and viral DNA (Heuer and Brown, 1997, 1998). They found several peptides involved in crosslinks to the C(2) position, and the 5′ end of the bottom strand [adjacent to the A(−2) position]; these peptides were localized to the catalytic core region. In contrast, a crosslink to the T5 position of a disintegration substrate contacted peptides in the C‐terminal, in the same 247–270 region as the T5 crosslink described here.
These data taken together provide clues to the important protein–DNA contacts required for integration. As would be expected, the catalytic core appears to bind the terminal LTR regions, at which the 3′ processing and strand‐transfer reactions occur. These regions also exhibit the highest level of sensitivity to mutation, and show the strongest protein–DNA crosslinking. The very strong crosslinks at the two terminal bases, coupled with previous data suggesting that these base pairs are likely to be disrupted upon binding suggests that these interactions may be critical for the proper alignment of the DNA in the active site. Gerton et al. (1998) recently suggested that Q148 might be involved in interactions with the 5 dinucleotide of the viral DNA. Our crosslinking data seem to confirm that this residue is capable of making close contact to the A(−2) position of the viral DNA. However, crosslinking at this position also occurred to Y143. Neither of these residues are visible in current crystal structures of retroviral integrases; they lie on the disordered loop just C‐terminal to the active site glutamate. This loop has been proposed to be involved in a conformational change that may occur upon DNA binding (Dyda et al., 1994). It is likely that once DNA binds to the active site, residues on this loop stabilize the 5′ end of the viral DNA. The fact that specific crosslinking to these two residues is seen also argues that the previously disordered loop probably becomes ordered upon DNA binding. It is not clear what the relative importance of the two crosslinked residues are. One caveat of this class of crosslinkers is that they have two separate electronic pathways for crosslinking. Crosslinks to aromatic amino acids occur by a higher energy triplet state, and produce much higher levels of crosslinking (Deitz et al., 1987). Therefore, it is possible that the Q148 interaction is as strong or stronger than that produced by Y143, but that it may produce lower signal intensity due to the preferred aromatic crosslinking pathway. One argument in favor of this is that a mutant in Y143 appeared to have little effect on 3′ processing or strand transfer (van den Ent et al., 1998), whereas a mutant in Q148 lowered levels of processing and strand transfer, and affected the choice of nucleophiles used by integrase (Gerton et al., 1998). However, more thorough mutagenesis is needed to rule out the involvement of either of these amino acids.
Crosslinking of the C(−1) position was isolated to a peptide spanning the region from 51–64. PTH derivative data were obtained for residues 51–60, but thereafter, signal was lost dramatically. This is most likely to be due to loss of the small peptide from the sequencing column matrix. Thus, the point of crosslinking is likely to be in one of the last few residues. W61 and Q62 are strongly conserved among retroviral integrases, and the positioning of Q62 in the active site region suggests that it may be involved in this interaction. Crosslinking to the aromatic W61 residue might also explain the rather strong signal observed at this position. A recent more detailed crystal structure of the core domain (Goldgur et al., 1998) provides a starting point for analyzing the likelihood of these predicted contacts. As shown in Figure 7, all of the residues potentially involved in the crosslinking are in the active site region. In addition, the spacing of these residues is consistent with the length of DNA involved; if the A(1) position binds in the vicinity of K159 and E152, then the C(−1) position interacting with Q62 and the A(−2) position with Q148 is well within the allowed spacing. The disordered loop has been traced in this structure, and Y143 is shown facing inward into the middle of the loop; however, the temperature factors for these residues are quite high, implying significant flexibility, so determining the exact positioning of Y143 and Q148 is difficult.
Structural summary of crosslinking data. The structure of the core domain of HIV‐1 integrase near the active site is shown in white, with residues discussed in this study highlighted in color. The three active site residues, D64, D116 and E152 are shown in red, while K159 is shown in blue. Q62, a strongly conserved residue on the peptide (51–64) which is crosslinked to the C(−1) position is shown in yellow. Y143 is shown in green, and Q148 in cyan.
Further upstream, in the proximal LTR region, there are clear interactions with the C‐terminal domain of integrase. Originally, it was believed that this region, which has a non‐specific DNA‐binding activity, was involved in target DNA binding (van Gent et al., 1991; Engelman et al., 1993). Recent results appear to rule this out; target specificity of chimeric integrases clearly aligns with the catalytic core region and not the C‐terminus (Shibagaki and Chow, 1997; Katzman and Sudol, 1998). These data, coupled with data from Heuer and Brown (1997), suggest that the C‐terminal domain is mainly involved in stabilizing the interaction of integrase with the viral DNA. This would explain the requirement for the C‐terminal domain for 3′ processing, but not for disintegration, which does not require any proximal LTR sequences (Balakrishnan and Jonsson, 1997). Stabilization of the proximal LTR sequence may be required in order to 'fit' DNA into the proper conformation to allow tighter binding of the terminal sequences; this could be accomplished by random interactions with DNA until the C‐terminal domain 'catches' a proper sequence in this region, whereupon the rest of the viral DNA is more tightly bound at the active site. The apparent lack of metal ion effects on these proximal positions argues in favor of a C‐terminal interaction; these regions are presumably far from the active site, and should be less influenced by the choice of metal cofactor.
Perhaps the most interesting finding is the region in the distal LTR which is both affected by mutation, and also crosslinks to integrase. This region exhibits a dramatic dependence on magnesium, which suggests that it is likely to be an interaction which involves the core domain. However, the levels of crosslinking are too low to have allowed identification of the exact region that is involved. It is unlikely that this region is contacting the same monomer of the core that is being contacted by the terminal LTR, which sits nearly 30 Å away. However, it is possible that this region is contacting the second monomer of the core found in the normal core dimer, or possibly even a completely different core dimer, since it is likely that the active complex includes at least two dimers of integrase. It is also important to note that this region, centered around position 9–10 is approximately one helical turn away from the region of terminal contacts, implying that both surfaces lie on the same face of the helix and may be contacted by adjacent integrase surfaces. These contacts are clearly vital in the presence of magnesium since mutation of positions 9–11 can nearly eliminate activity. It is not clear, however, whether these same contacts are required in vivo to the same extent. Some data seem to suggest that DNA past positions 6–7 are not necessary in vivo; however, there are several reports of mutations at other regions of the LTR which appear to affect viral replication. For instance, in Moloney murine leukemia virus, a mutation at positions 10 and 11 inhibits viral replication, and appears to suffer a defect in 3′ processing (Murphy et al., 1993). It is also possible that other retroviral integrases use slightly different patterns of DNA recognition. In the case of FIV integrase, the C‐terminal domain appears to not be required for 3′ processing activity (Shibagaki et al., 1997), suggesting that the stabilization by the HIV C‐terminus may be carried out by a different portion of the protein, or may not be necessary at all.
Finally, there is the question of strand‐transfer activity. Once cleavage occurs, strand transfer is carried out, presumably using the same active site that was used for the cleavage reaction. Strand transfer has previously been suggested to require much less sequence specificity than 3′ processing (Balakrishnan and Jonsson, 1997). Our data confirm that strand transfer with precleaved substrates does not appear to be affected by sequences upstream of position 3, in the presence of either cation, while mutations at positions 1 and 2 show a significant effect on levels of strand transfer (data not shown). One explanation for this is that the DNA must be bound in a more specific confirmation for the 3′ processing reaction than for strand transfer; thus binding of the upstream regions may no longer be required for the interactions involved in the integration step. This may suggest that once 3′ processing occurs, there is a slight change in the orientation or positioning of the DNA in the active site which helps to position the DNA for strand transfer. However, the C‐terminus of integrase is still required for strand transfer, although there is no evidence that it carries out the same role as it does in 3′ processing.
Further determination of the exact contacts between the crosslinked DNA and integrase will help to locate precisely the position of the LTR on the protein. It is known that lysine 159, near the active site, is involved in interactions which may orient the DNA properly for cleavage (Jenkins et al., 1997). It is possible that a similar orientation of viral DNA occurs upon binding of T(5) to the C‐terminal domain, perhaps in the large cleft seen in the structure of that region. Isolation of the residues which contact the T(5) position may also help us to connect the structures of the C‐terminal domain and the core to produce a clearer picture of the complete integrase–DNA complex.
Full‐length HIV‐1 integrase (F185K/C280S) was expressed in E.coli and purified from soluble extracts. Integrase contained a histidine tag at the N‐terminus of the protein to facilitate purification. A thrombin cleavage site was engineered adjacent to the histidine tag to allow removal of the tag after purification. After sonication and preparation of a soluble extract, integrase was purified over a Ni‐affinity column, incubated with thrombin to cleave the tag, and then purified over a benzamidine Sepharose column to remove thrombin. The protein was further purified over MonoS to chromatographic homogeneity as visualized by Coommassie Blue stain. The details of these purification procedures have been previously described (Jenkins et al., 1996). Final integrase samples were dialyzed into buffer containing 1 M NaCl and 50 μM zinc acetate. Concentrations were determined using calculated extinction coefficients based on amino acid composition. In order to reduce variability in specific activity, purified integrase was divided into separate aliquots and frozen in liquid nitrogen; aliquots were used only once, and any remaining protein was discarded. All experiments in this study were carried out using integrase from a single purification.
Oligonucleotides containing mutations in the LTR sequence were synthesized on a Millipore Expedite DNA synthesizer at the 40 nmol scale. After deprotection at 55°C in ammonium hydroxide for 8 h, samples were dried down, resuspended in 10 mM Tris–HCl pH 8.0, and ethanol precipitated. The oligonucleotides were then gel purified on a 20% denaturing acrylamide gel, passively eluted from the gel, and ethanol precipitated. These oligonucleotides were completely pure as judged by chromatography of samples labeled with [γ‐32P]ATP. For oligonucleotides containing a 3′ biotin tag, synthesis was carried out on a Biotin‐TEG support matrix (Glen Research Inc.). Oligonucleotides containing 5‐iododeoxyuracil or 5‐iododeoxycytidine from Glen Research were synthesized using normal protocols, except that deprotection was carried out at room temperature for 24 h. Substrates for 3′ processing and strand‐transfer reactions were made by labeling the top strand oligonucleotide (containing the 3′ biotin tag) at the 5′ end with [γ‐32P]ATP (3000 Ci/mmol) using T4 polynucleotide kinase, and annealing the labeled primer with the bottom strand oligonucleotide as previously described (Jenkins et al., 1997). Annealed substrates were purified over a Bio‐Rad P6 column. Substrates for photocrosslinking were made in the same manner, except that the strand containing the photoactive residue was always labeled.
Manganese (Mn)‐based 3′ processing reactions contained 25 mM 3‐morpholinopropanesulfonic acid (MOPS) pH 7.2, 10 mM β‐mercaptoethanol, 10% (v/v) glycerol, 100 μg/ml bovine serum albumin, 10 mM MnCl2, 5 nM DNA substrate, and 1 μM integrase in a final volume of 100 μl. NaCl was added to a final concentration of 50 mM including contributions from the protein. The magnesium (Mg)‐based 3′ processing reactions contained 25 mM 3‐morpholinopropanesulfonic acid (MOPS) pH 7.2, 5% (v/v) PEG‐8000, 5% (v/v) dimethylsulfoxide, 10 mM DTT, 10 mM MgCl2, 15 mM potassium glutamate, 5 nM DNA substrate, and 1 μM integrase in a final volume of 100 μl. An additional 30 mM NaCl was introduced into these reactions with the protein. In both cases, reactions were incubated for varying amounts of time, and 10 μl aliquots were removed, stopped with 5 μl loading dye, and electrophoresed on NuPage 4–12% MES gels (Novex, Inc.). Quantitation of reaction products was carried out using a PhosphorImager and ImageQuant 4.2 software (Molecular Dynamics).
Selection substrates were constructed by mixing oligonucleotide DOM‐155 (5′‐CAGCTTCGTCGCAGGTCAG) with one of the bottom strand degenerate oligonucleotides and annealing by heating to 95°C for 5 min followed by slow cooling to room temperature. Bottom strand primers had previously been phosphorylated at their 5′ ends with T4 polynucleotide kinase and unlabeled ATP. The top strand of the annealed duplex was extended with the Klenow fragment of E.coli DNA polymerase, and the duplex substrate was purified over a Bio‐Rad P‐6 desalting column. Standard 3′processing assays were carried out under Mg or Mn conditions as described above, using 5 nM duplex substrate, for 2 h at 37 °C. This sample was then ethanol precipitated, and resuspended in 10 mM Tris–HCl pH 8.0 (20 μl). The LM–PCR linker was constructed by mixing oligonucleotides DOM‐152 (5′‐GTATGCACGATG) and DOM‐153 (5′‐CCCGTGACCGTGAGCATCGTGCAT), and annealing by heating to 95°C for 5 min followed by slow cooling to room temperature. A quarter of the reaction mixture was added to 0.7 μM annealed linker in 1× T4 DNA ligase buffer and 10% PEG‐8000. Ten units of T4 DNA ligase were added to the total volume of 20 μl, and ligation was carried out for 2 h at 25 °C. The sample was diluted to 50 μl with water, and desalted over a Bio‐Rad P‐6 column. A small sample (1 μl) of the ligated product was used as template in a 50 μl PCR containing 1× PCR buffer, 2 mM MgCl, 100 μM dNTPs, 100 ng flanking primers, and 2.5 U Taq polymerase. After 25 cycles of PCR, the samples were digested with EcoRI and HindIII, purified over Wizard columns (Promega), and ligated to pUC18 linearized with EcoRI and HindIII. The ligated DNA was transformed into E.coli DH5α, and white colonies were picked and grown overnight in LB broth. DNA was prepared using the Qiagen Turbo method on a BioRobot 9600 (Qiagen, Inc.), and sequenced on an ABI 391 sequencer using the manufacturer's protocols (PE Biosystems). Two separate primers were used to sequence the inserts; one from the top strand on the left side of the pUC polylinker, and one on the opposite strand from the right side. Sequence analysis was carried out with EditView (PE Biosystems) and MacDNASIS (Hitachi Software).
Reaction mixtures contained the same components as the 3′ processing reaction, with 5 nM DNA substrate and 1 μM integrase in a final volume of 5 ml. Samples were incubated for 15 min on ice, and transferred to a 96‐well microtiter plate and irradiated with 302 nm light from a handheld ultraviolet lamp with a mean flux of 2.0 mW/cm2 for 35 min. Samples were then centrifuged at 16 000 g for 10 min, and the pellet resuspended in buffer containing 25 mM HEPES pH 7.5, 10 mM EDTA, 10 mM DTT, and 6 M urea, boiled for 5 min, and electrophoresed on a NuPage 4–12% MES gel (Novex, Inc.). The gel was autoradiographed, and portions corresponding to crosslinked product were excised, crushed in buffer containing 20 mM Tris–HCl pH 8.5 and 0.5% SDS, and allowed to elute passively overnight at 37°C. Recovery of labeled counts from this procedure was quantitative. Protein–DNA complexes were precipitated with 10% trichloroacetic acid (TCA), washed with acetone, dried, and resuspended in protease buffer. For cleavage with cyanogen bromide (CNBr), the samples were resuspended in 100 μl 70% trifluoroacetic acid, and 100 mM CNBr was added, followed by incubation for 45 min at 70°C. These samples were dried down, and resuspended in NuPage gel loading buffer (Novex Inc.). For cleavage with endoproteinase Glu‐C, samples were resuspended in 50 mM Tris–HCl pH 8.5, 5 mM EDTA, 5 mM DTT, 1 M NaCl and 0.1% SDS, and 200 μg Glu–C (Boehringer Manheim) were added per microgram of integrase and incubated for 12 h at 37°C. Samples were precipitated with TCA and acetone, dried, and resuspended in gel loading buffer. The peptides were then electrophoresed on NuPage 4–12% MES gels, and transferred to PVDF membranes (Trans‐Blot, Bio‐Rad Laboratories) using a Novex blotting apparatus. Radioactive peptides were identified by autoradiography, excised from the membrane, and analyzed on an HP 241 protein sequencer (Hewlett‐Packard).
We thank Dr Tanya Paull for careful reading of the manuscript, and Dr Dale Ramsden for advice on the LM–PCR selection experiments. Molecular graphics images were produced using the MidasPlus program from the Computer Graphics Laboratory, University of California, San Francisco. This work was supported by the National Institutes of Health Intramural AIDS Targeted Antiviral Program.
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text:
Art Blooms Eternal
As Natick's inaugural artist-in-residence, Kira Seamon has so far been the epitome of a good neighbor.
And while the role marks a first for both the town and artist, it's likely some Natick residents have heard of Seamon before, and perhaps crossed paths with some of her creations.
Last year, she took part in group exhibits at the Morse Institute and Bacon Free libraries, the latter of which just ended in September. Seamon will return to the Morse Institute Library in May and June of next year in an exhibit featuring her photography of Peru.
Seamon also displayed her "Stained Glass Sky" kaleidoscopic photography last year at the Morse Institute Library. The images consist of natural subjects - trees, flowers, skylines - and are filtered and captured through a special lens. The result is a striking kaleidoscopic effect that challenges the viewer to decipher the subject matter's origins.
Sponsored by the Natick Center Cultural District, the role of artist-in-residence entails being an active advocate for the arts, and includes a stipend to help facilitate that work. Studio space is also provided as a perk of the program, and the artist-in-residence is expected to organize and host several exhibitions throughout his or her tenure.
Seamon is a Dedham resident, and first learned about Natick's plan to host an artist-in-residence during her exhibit at the Bacon Free Library. A librarian there suggested she apply for the program, and the rest is art history.
"I have an artistic second home in Natick," said Seamon.
Since April of this year, Seamon has been promoting and creating art in the town as part of her tenure. She has played a role in Natick Days, the town's annual community festival.
After the recent fire in downtown Natick, Seamon organized and produced an artistic show of gratitude for emergency crews that responded to the crisis. Several posters were filled with the traced hands of volunteers, and the finished works presented to the Natick Fire Station for display.
Also as part of her artist-in-residence role, Seamon is organizing an "Art In Bloom" event at the Morse Institute Library, which will be held on Sunday afternoon, November 17th between 1:00 and 5:00.
Art In Bloom is a celebration of existing works of art, rendered in themed, floral arrangements. Seamon has extensive experience with these horticultural homages, having hosted and assembled many iterations of the exhibit over the years.
Prior exhibitions of Art In Bloom have included tabletop displays of flowers, each an interpretation of a well-known and beloved book. Nathaniel Hawthorne's "The Scarlet Letter" was one of these, with its titular, capital letter "A" represented in red carnations within the backdrop of varied, violet flowers.
Another, a nod to Alice Walker's "The Color Purple," features the book in a blossom of flowers of that same hue, a celebration of sorts, of the novel's life and influence. It's quite literally (literary) art imitating art.
For this month's version of Art In Bloom, the floral arrangements will represent Seamon's own photography as centerpieces and inspiration, including some recent images from her excursion to Peru.
The Natick Garden Club will lend their expertise and furnish flowers for the arrangements. In addition, musicians from Natick's Walnut Hill School will perform some classical pieces during the event, between the hours of 2:30 and 3:30.
Seamon's interest and involvement in Art In Bloom began with her exposure to the idea years ago, when she attended exhibitions of the same name hosted by the Museum of Fine Arts.
A self-described superfan of Art In Bloom, she soon began organizing and hosting her own versions of the event throughout the state, mostly in libraries.
Seamon's favored mediums will culminate in this month's Art In Bloom event, featuring floral arrangements as odes to her photography. The November 17th exhibit will highlight her kaleidoscopic photographs, as well as several images the artist captured during a recent trip to Peru.
"It's truly beyond a dream come true," said Seamon. "And I hope to give my all to the Natick community."

redpajama_set_name: RedPajamaCommonCrawl


text:
Lady Lake es un pueblo ubicado en el condado de Lake en el estado estadounidense de Florida. En el Censo de 2010 tenía una población de 13.926 habitantes y una densidad poblacional de 643,09 personas por km².

Geografía
Lady Lake se encuentra ubicado en las coordenadas . Según la Oficina del Censo de los Estados Unidos, Lady Lake tiene una superficie total de 21.65 km², de la cual 20.91 km² corresponden a tierra firme y (3.44%) 0.75 km² es agua.

Demografía
Según el censo de 2010, había 13.926 personas residiendo en Lady Lake. La densidad de población era de 643,09 hab./km². De los 13.926 habitantes, Lady Lake estaba compuesto por el 90.82% blancos, el 5.09% eran afroamericanos, el 0.35% eran amerindios, el 1.08% eran asiáticos, el 0.09% eran isleños del Pacífico, el 1.31% eran de otras razas y el 1.26% pertenecían a dos o más razas. Del total de la población el 4.27% eran hispanos o latinos de cualquier raza.

Referencias

Enlaces externos

Pueblos de Florida
Localidades del condado de Lake (Florida)

redpajama_set_name: RedPajamaWikipedia


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redpajama_set_name: RedPajamaGithub


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Have a glimpse of some of the caves below. There are actually a lot more!
Here is where the ikat cloth, the oldest burial cloth in Southeast Asia now displayed in the National Museum, was found. Situated in a cliff facing the rising sun, it is a small cave which is very difficult to reach. Its name means bird's manure in the local language as it is the dwelling place of many swifts (balinsasadaw). Skulls, bones and some intact Banton coffins (small-sized coffins made of hard wood with handles shaped in the likeness of animals) remain there.
This is the biggest cave re-discovered so far named so because of its size. It can house probably a hundred people. There were no bones and skulls found and initial observations suggest that the cave was used as a dwelling place of pre-historic people but could have been used also as a hiding place of the townsfolk during the war.
This cave is situated at the top of a mountain and believed used as dwelling place as suggested by the seashells, bones of edible animals and pieces of pottery found here. It seemed to be a perfect location as a hideout, given its narrow and difficult vertical entrance. It can easily be camouflaged by covering the entrance with a one-and-a-half-foot diameter rock. There is another entrance at the top but it is more difficult to find and reach. It has one chamber. Beside it is a small flat terrain – a perfect gathering place.
This cave is a very shallow one, in fact, it is as if it is just sliced (tapyas) or carved out of a huge rock wall, hence the name. At its foot were excavated human remains that seemed to be hollowly buried. As such, it is believed that this cave is a site for preliminary burials.
This is another burial cave. It is named after the smooth diagonal-surfaced rock where one can practically slide from its mouth to the stones below.
This one's named so because it perfectly resembles a tent. This is a beautiful cave but so far, no historical remains have been found.
This cave is one of the easiest caves to reach and the first one that got a name. The cave got its name from the abundance of sunshine that gets into it through its mouth facing the rising sun. Besides, it was named by the people behind the Silak publication (Lyndon Fadri, Ismael Fabicon, Abner Faminiano and Rodil Fadri).
In the cave are skulls and bones of primitive inhabitants. This cave offers a breath-taking view of the Togbongan coastline.


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High School Senior Uses Yearbook Quote to Make the Perfect Joke About His Name
Leul Dadi tweeted that after 18 years, his last name had finally paid off.
By Marissa Miller
Twitter / @LeulDadi
Graduation tends to reveal everyone's true colors. Sometimes you learn your place of education can't spell its own name, and sometimes you realize you haven't given your fellow classmates nearly enough credit for being hysterical. The internet goes nuts for students who leave a lighthearted lasting legacy, and that's precisely what happened after one student shared his epic quote.
Dallas-based user @LeulDadi's tweet is going viral because it seems like he had been waiting all his life to use his last name in this context.
There's no word on whether he is asking the two girls below and above him to call him "Dadi," but it just so happens that he seems to be pretty cozy with the one above. They'd make a cute prom king and queen, TBH:
Judging by the replies, it seems like the entire school got into the spirit. Including the faculty.
The comments demonstrated mixed reactions to the three students' quotes shown in the tweet, but an overwhelming majority seems to favor Leul's, which is short, sweet and hilarious. Keep that in mind when you're asked to craft one of your own next year.
Related: The 'Dear White People' Cast Opens Up About the Show's Importance
Keywordsyearbookyearbook quoteshigh school


managed. Tenants are carefully screened and asked to meet a standard set of requirements.
housing opportunities by contacting the organizations listed at the end of this document.
difference between that and the cost of the apartment.
based on the HUD guidelines. Contact your local housing authority for more information.
income households, and the rest of the building at market rate (usually without rent control).
about any problems that you have with credit and the steps you have taken to address them.
and can demonstrate ability to pay the rent.
If you or someone you know needs help with credit, contact a credit counseling agency.
deposit or provide a co-signer in lieu of a positive credit report.
to accept a deposit in installments.
a. Call a specific property when a waitlist opens and ask.
the income maximums for the housing you are applying for.
Make a list of every city or community where you would consider moving.
• Senior: You or your spouse must be at least 62 or older.
• Special Needs: You must have a disability (mental, physical, development).
but Delancey Street is also a good option for resources about housing and services.
Call or visit the website.
Check the website of non-profit developers at least monthly.
properties you have an interest in and are qualified for.
or go to the property to get an application, complete it, and submit it by the deadline.
managed by other affordable housing companies.
Apply to as man non-profit property management companies as you can.
which you want to live. Many owners advertise on site.
contain the greatest number of new listings. Also, try out craigslist.org for apartments.
415-928-5910 465 S. First St.
1001 Willow St. Eden Housing Inc.
Chico, CA 95928 409 Jackson St.
Santa Rosa, CA 95404 1017 Main St.
Oakland, CA 94607 Housing for Independent People, Inc.
Oakland, CA 94602 Human Investment Project, Inc.
510-247-2448 364 South Railroad Ave.
Fresno, CA 93721 Satellite Housing, Inc.
Petaluma, CA 94952 230 Fourth St.
Roseville, CA 95828 201 Eddy St.


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Maine man's letters from jail net 9-month sentence
AUGUSTA (AP) — Authorities say a man serving a nine-month jail sentence sent 16 letters from behind bars to his 1-year-old daughter, but the messages were really for the little girl's mom.
Prosecutors said the letters from Robert Vincent Johnson told the woman to avoid cooperating with the district attorney. The 30-year-old Johnson said if authorities couldn't find the woman, domestic violence charges against him would be dismissed.
The Kennebec Journal (http://bit.ly/1i0QZBG ) reported that prosecutors say he was trying to urge the woman to withhold information.
Johnson, of the Madison/Skowhegan area, was sentenced to nine months in jail this week after pleading guilty to a charge of tampering with a victim and violating conditions of release.
Johnson's attorney said her client was remorseful and ashamed of his actions.
jail, letters, Maine, tampering


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DAY6 is ready to rock with MYDAYs in 'Gravity' Concert in Singapore
The rising rock band, DAY6, returned to our sunny island for the third time! The 5-membered boy band held their world tour concert, 'GRAVITY' at The Star Theatre on 5 October 2019, Saturday.
The show started off with a bang as DAY6 entered the stage with "Best Part", which left fans on their feet, jumping at their seats and jamming to the music. The packed venue was beautifully lit up with a sea of fanlights My Days (affectionate name for DAY6 fans) were holding, creating a truly beautiful sight!
Mediocre certainly does not exist in DAY6's dictionary! Contrary to their goofy and witty personality offstage, DAY6 professionally delivered high-quality performances throughout the show. My Days in Singapore were in for a treat with the explosive tracks, "Sing Me", "I Wish", "Somehow" and their latest song, "Time of Our Life". The vibes at the venue were crazy! On top of the exceptionally intense lighting and stage effects, the voices of My Days singing along to the hit tracks of the band undeniably gave everyone goosebumps.
[#DAY6inSG] Now it's 'Time of Our Life' !! #DAY6GRAVITYinSG #GravityinSG pic.twitter.com/0ZxwfcSQi8
— GOKPOP (@goKPOP_Asia) October 5, 2019
The cool electric guitarist, Jae, teased the fans that he read the tweets from My Days who expressed their longing to hear the tracks from the band's younger days. "Because of your amazing and endless stream of tweets, we have changed the setlist to the songs that you guys want to hear," revealed Jae who communicated to fans in fluent English throughout the concert. Of course, when it comes to fan service, Jae surpasses everyone else, right?
The atmosphere was intensely hyped up as the experienced band hooked fans with "So Cool", "Out of My Mind", "Feeling Good", "How to Love" and "For Me". The moment all fans were anticipating for was none other than the track, "Wanna Go Back", where the adored drummer, Dowoon, sings. DAY6 could not hold back their excitement to hype the youngest of the band during his part in the song, leaving My Days exclaiming with glee at the adorable sight!
The surprises surely did not come to an end! The concert continued with DAY6 performing a refreshing medley of not only their songs but also western hit tracks—"Shape of You" , "Like That Sun", "Days Gone By", "Treasure", "Blood" and "Get Lucky", assuring My Days the three-year-wait was worthwhile. The band also had a throwback to their old but gold tracks, "Colours", "I Need Somebody", "I Wait" and "Lean on Me".
[#DAY6inSG] Throwback to the Every DAY6 Project, it's time for 'I Need Somebody'#DAY6GRAVITYinSG #GravityinSG pic.twitter.com/h0ldzQvpno
The highlight of the concert was undeniably the powerful solo stages. Witnessing Dowoon on his drums, Young K on his bass, Sungjin on his guitar, Wonpil with his synth and Jae on his electric guitar, DAY6 proved their unquestionable musicianship as they flawlessly slayed the stage.
Like idol like fan— DAY6 performed with little to no breaks yet they were constantly full of energy. The continuous roaring proved that My Days were certainly of no exception! Regrettably, nothing gold can stay, the band left the stage with bass-strong tracks like "Warning!" and "Shoot Me", leaving fans with an entertaining VCR.
[#DAY6inSG] BANG BANG ❗ DAY6 with 'Shoot Me'#DAY6GRAVITYinSG #GravityinSG pic.twitter.com/NtqTK9HHq0
DAY6 united back onto the stage for the encore with "Cover". Passionate My Days were singing along with the boys as everyone waved their mobile flashlights along to the soothing track. My Days in Singapore even prepared a tear-jerking video for the band, which highlighted the journey and achievements of DAY6. It was a touching sight as My Days sang along to "Beautiful Feeling" the BGM of the video.
After the emotional video made by the loving My Days, DAY6 was visibly touched and were speechless for a while, being closed to tears. Feeling overwhelmed, Jae signaled for the members to convey their thoughts and feelings one at a time. Wonpil shared, "I really felt goosebumps. Firstly, this is our first time doing a concert in Singapore, I know everyone is busy but thank you for taking your time off to come here with us." He also commented that Singapore has a lot of delicious food which he enjoyed very much. "I hope everyone had a great time today. Please always be energised and I hope you guys will only hear and see good things, thank you for giving us unforgettable memories. I just hope everyone is happy, thank you everyone. We will be here next time."
The suave bassist went on with his ment in English. "You guys are my motivation, you inspire me and you guys are the reason why I sing, I write songs, and go on, so thank you very much. You guys are telling me that we're doing well right? Honestly, we, DAY6 have this many people telling us that we're doing well. It might not be enough for you guys but we want to tell you that you are doing your best, you are doing well so you have the right to be happy so we are going to wish you all the happiness that you can. Thank you very much, we hope that you are always happy. Eat a lot and stay healthy."
Our beloved drummer, Dowoon, expressed, "The last time we were here for a fanmeeting and now we're here for a concert, thank you for allowing us to be on this stage once again. I am really happy today, you guys touched my heart. (he said this in English!). We will be back, we will come back, yeah. Happiness! Be happy everyone." As expected from our vibrant and bubbly youngest member of the band, My Days squealed with delight at Dowoon's adorable remark.
"Singapore My Days' voices are so beautiful, (you guys) touched my heart, thank you. Work hard, play hard and study hard, on the stage, I'll do my best (amazing English, as expected from Bob!). I'm still lacking so I will work harder to be someone you can be proud of, thank you so much guys," stated the leader of the band, Sungjin, who sweetly did his best to communicate to the fans in English. That's our leader!
Jae, being the smooth-talker he is, expressed in English, "Today could not have been more beautiful, more perfect, energetic, you guys were crazy today, Singapore goes HARD! I really appreciate you guys for sharing the energy with us, thank you for heeding the instructions, though looking a little dumb with us (jumping along to the music), we hope you all had as much fun as we had today. Honestly thank you very much for having us back here, hopefully see you all soon!" The concert ended on a high note with DAY6 performing the classics, "You Were Beautiful", "Congratulations" and "Dance Dance" before officially closing the concert with "Freely".
[#DAY6inSG] Debut track!! DAY6 is back on stage with 'Congratulations' !! 💙#DAY6GRAVITYinSG #GravityinSG pic.twitter.com/us0OlBrN08
DAY6, who showed deep sincerity and professionalism throughout the show, left the stage with a final group bow towards My Days. The concert was undoubtedly a special one for My Days as they could not bear to part with the boys and conclude the amazing night.
I guess we can confidently say that the jam-packed concert was a blast and DAY6 surely made 'My Day' a memorable one! On a side note, DAY6 will be making a comeback with "The Book of Us: Entropy" on 22 October. Do show your support for their upcoming comeback!
We would like to extend our gratitude to Live Nation Singapore for the invitation.
Written by: Amanda Chin
Official photos by: Live Nation Singapore
Follow our social media platforms for the latest K-wave news
Twitter | Instagram | Facebook
Tags: Day 6 World Tour Gravity in Singapore, day6, eventcoverage, eventcoveragesingapore
GOKpop Singapore


You may not be able to kick someone else's afterward, but you can kick up to 450 calories off your own tail in an hour with Tosha Jeffries.
Jeffries, a 27-year-old mother of two, guides her classes through plyometric and calisthenics-based moves designed to warm up and stretch muscles that will be worked with fat-burning combinations of jabs, crosses, knee drives and kicks before a cool down that incorporates elements of Pilates and yoga. The result is 15 sweaty bodies happy that they can still make it to the door under their own power when class is over.
Fitness experts credit cardio kickboxing workouts with not only burning calories but with letting participants vent their daily stress, keeping the heart in shape, and maintaining speed, strength, balance and agility.
Jeffries, who has been teaching for nine years, said she enjoys the variety of the workouts and her students agree.
Teresi, a dancer in college who has been in the class for about two months, said Jeffries "kills us with squats a lot" and added that her college experience isn't that much help in class.
"I think she uses a lot of different muscles that I'm not used to," she said.
Jeffries, who has taught at several Rockford-area gyms, said she has worked over the years to keep the classes fresh.
Maxson followed Jeffries' class from another gym and has been a participant for about year despite a motorcycle accident that required knee surgery.
Know your level of fitness and look for a class that accommodates various skill levels.
Warm up and stretch before each session, concentrating on shoulders, including the rotator cuff; quadriceps, hamstrings, glutes and calves.
Wear comfortable clothing that allows you to move your arms and legs easily.
To avoid putting too much stress on your joints, don't lock out the elbows on the punches or the knees on the kicks.
Wear shoes that support the heel and the metatarsal areas of the foot.
Pay attention to foot positioning on punches; some twisting punches (such as a cross) require pivoting on the appropriate foot to avoid placing too much of the twisting motion on the knees.
Work at your own pace and rest as needed.
Drink plenty of water before, during and after the workout.
Follow each session with light cool down exercises and stretches.
Sources: healthatoz.com, kidshealth.org and womenfitness.net.


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Home>Senior Living
Helping a Senior with Stroke Recovery
If you're currently a caregiver for a senior that has suffered a stroke, your role in providing them with the support they need can make a big difference in keeping them safe at home. Here are five tips to help make that happen and assist them with their stroke recovery.
By First In Care|2021-02-16T16:51:06-05:00May 17th, 2021|Categories: Senior Living|Tags: caregiver assistance, Senior Care, senior health, stroke, wellbeing|
Recognizing the Signs of a Stroke in Seniors
Nearly three-quarters of all strokes occur in people over age 65, and the risk of stroke more than doubles each decade after the age of 55. The risk for strokes in seniors is concerning and these are sobering statistics for them and their caregivers. Here are some of the signs to look for so you can get them the crucial early treatment they need.
By First In Care|2021-02-16T16:18:34-05:00May 3rd, 2021|Categories: Senior Living|Tags: caregiver assistance, Senior Care, senior health, stroke|
What to Expect After a Senior Experiences a Stroke
Millions of Americans suffer a stroke every year. If you are currently caring for an aging in place elderly loved one who just had a stroke, here's what to expect after they leave the hospital.
By First In Care|2020-10-31T00:51:52-04:00November 2nd, 2020|Categories: Senior Living|Tags: caregiver assistance, mini stroke, senior health, stroke|
Why is Proper Nutrition So Important for Aging in Place Seniors?
Some of the main reasons why elders get malnourished include depression, impaired dentition and a lack of nutritious food in the home. For those seniors who are aging in place proper nutrition is so important for maintaining their quality of life and independence.
By First In Care|2020-10-15T12:57:27-04:00October 26th, 2020|Categories: Senior Living|Tags: Dietary Tips, Meal Preparation, nutritional needs, senior nutrition, Senior's Diet|
How to Make Sure Seniors are Prepared for Hurricane Season
When a hurricane makes landfall, residents aged 65-and-over are most vulnerable because of health problems, diminished awareness and transportation challenges. In the storm's aftermath a lack of food, water, shelter and medicine can quickly place the elderly at risk. If your aging in place parents currently live near the coast here's how to help them prepare for the next storm.
By First In Care|2020-09-07T16:10:43-04:00September 8th, 2020|Categories: Senior Living|Tags: Disaster preparedness, hurricane preparedness, hurricane season, natural disasters, Senior Safety|
5 Low-Impact Exercises for Seniors
Many older adults suffer from arthritis and reduced bone density that limit their strength, flexibility, and balance. That makes finding effective forms of exercise for seniors that don't place a lot of stress on their bodies a priority. If that's a concern for you, here are 5 low-impact workouts that most seniors can use to stay fit.
By First In Care|2019-10-04T12:49:32-04:00October 7th, 2019|Categories: Senior Living|Tags: arthritis, balance, Exercise, senior health, water aerobics|
How Can I Help My Elderly Parent Sell Their Car?
Helping an elderly parent sell their car can be challenging and stressful if you haven't done it before. There are several steps that will need to be taken to ensure that the selling process goes smoothly, and that you're getting mom a fair price for her vehicle. To help you develop a more effective car selling strategy, use these tips from the pros.
By First In Care|2019-07-07T11:56:55-04:00July 8th, 2019|Categories: Senior Living|Tags: car, sell car, Transportation, vehicle|
The Benefits of Water Aerobics for Seniors with Arthritis
Regular exercise is a great way to help keep an aging in place senior healthier, happier and more independent. But for the millions of seniors with arthritis, getting the exercise they need is oftentimes a daily struggle. If you're a caregiver for an aging in place loved one with arthritis, getting them to a pool nearby that offers water aerobics for seniors will help ensure that they get the exercise their body so desperately needs.
By First In Care|2019-06-24T07:33:03-04:00June 24th, 2019|Categories: Senior Living|Tags: arthritis, Exercise, mobility, stress relief, water aerobics|
How SilverSneakers is Helping Caregivers Keep Seniors More Active
Regular exercise benefits a senior in so many ways, including improving balance, flexibility and coordination, strengthening their immune system, building lean muscle, reducing stress and promoting a better night's sleep. If you're looking after a senior in the Manatee County, FL, area, one of the exciting new ways you can help keep them more active is by introducing them to the SilverSneakers program.
By First In Care|2019-06-08T14:45:57-04:00June 10th, 2019|Categories: Senior Living|Tags: Active, balance, Exercise, health, SilverSneakers, wellbeing|


Current interest rate is based off of New York Prime of 3.25% with a floor of 2%. Wichita County is an equal opportunity lender.
The Neighborhood Revitalization Plan offers tax rebates for 10 years for projects that increase your properties assessed value at least $5,000. Applications must be turned in before the project begins. Applications can be obtained in the Wichita County Appraisers' Office or in the Wichita County Economic Development office.
The purpose of this program is to improve the appearance and functionality of commercial properties located in Wichita County. Improving the appearance and functionality of commercial buildings is part of an overall strategy to attract consumers and new businesses as well as to support existing businesses in Wichita County.


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Charly Wagner (eigentlich Karl Heinz Wagner, * 8. Juni 1941 in Köln; † 7. Mai 2020 ebenda) war ein deutscher Radio-Journalist und Moderator.

Leben
Charly Wagner trat schon in seiner Jugend im Rundfunk auf, als er im September 1953 am Hörspiel Ich kannte die Stimme mitwirkte. Er absolvierte eine Ausbildung zum Versicherungskaufmann, bevor er nach einer Sprecherschulung bei der Deutschen Welle und anschließender Arbeit als Redakteur bei dieser ab 1965 als Nachrichtensprecher und Ansager vom WDR fest angestellt wurde. Ab 1973 war er im Hörfunk auch als Moderator eigener Sendungen tätig, so gemeinsam mit Max Schautzer bei Daheim und unterwegs. Seit den 1980er Jahren arbeitete er auch als Ansager für das WDR Fernsehen. Mit Sendungen wie Musikpavillon, Swing und Balladen, Orchester der Welt und Schellack-Schätzchen wurde er "die Stimme" von WDR 4.

Nach 39 Jahren beim WDR ging Wagner im Jahr 2004 in den Ruhestand. Er moderierte bis 2006 Swing und Balladen und kehrte später für vereinzelte Auftritte als Vorleser in der Rubrik Klassiker des Herrenwitzes in die Harald Schmidt Show zurück.

Wagner starb im Alter von 78 Jahren in seiner Heimatstadt Köln. Er wurde am 5. Juni 2020 auf dem Friedhof Lechenich in einer Urne beigesetzt.

Weblinks
 Berichte von Charly: Eine Sammlung charmanter Versprecher bei wdr.de
 , Charly Wagner in der Harald Schmidt Show, hochgeladen am 21. August 2014

Einzelnachweise

Hörfunkmoderator
Hörfunkjournalist
Journalist (Deutschland)
Nachrichtensprecher
Person (Westdeutscher Rundfunk)
Person (Medien, Köln)
Deutscher
Geboren 1941
Gestorben 2020
Mann