extfieldsofstudy: Biology Medicine

s2fieldsofstudy: Biology Medicine

text:
Long-lived cytotoxic T lymphocyte memory in mucosal tissues after mucosal but not systemic immunization.

The induction and maintenance of long-term CTL memory at mucosal surfaces may be a critical component of protection against mucosal pathogens and is one goal towards development of effective mucosal vaccines. In these studies we have functionally evaluated short and long-term CTL memory in systemic and respiratory or genital-associated lymphoid tissues following mucosal or systemic routes of immunization. Our results indicate that shortly after immunizing mice with a recombinant adenovirus vector expressing glycoprotein B (gB) of herpes simplex virus (AdgB8), gB-specific CTL memory responses were observed in systemic and mucosal immune compartments regardless of the route of inoculation. In contrast, several months after immunization, anamnestic CTL responses compartmentalized exclusively to mucosal or systemic lymphoid tissues after mucosal or systemic immunization, respectively. Furthermore, the compartmentalized CTL memory responses in mucosal tissues were functionally observed for longer than 1.5 yr after intranasal immunization, and CTL precursor frequencies one year after immunization were comparable to those seen shortly after immunization. Therefore, to our knowledge, this is the first functional demonstration that the maintenance of anti-viral memory CTL in mucosal tissues is dependent on the route of immunization and the time of assessment. These results have important implications for our understanding of the development, maintenance, and compartmentalization of functional T cell memory and the development and evaluation of vaccines for mucosal pathogens, such as HSV and HIV.

M emory is a hallmark of both humoral and T cellmediated immune responses and is typified by a more rapid and intense immune response on re-exposure to the same or closely related antigen. Indeed, a desirable property of vaccines is the ability to generate long-term immunological memory capable of preventing infection or limiting disease. Our current understanding ofT cell memory stems from studies of systemic immune responses, however little is known concerning the induction and maintenance ofT cell memory responses in mucosal tissues. Numerous pathogens, including respiratory, gastrointestinal and sexually transmitted agents, such as HSV and human immunodeficiency virus (HIV), initiate infection at mucosal surfaces. Since the mucosal immune system is somewhat separate and distinct from systemic immunity (1-3), a better understanding of specific immunologic T cell memory in mucosal tissues should contribute to the development of effective mucosal vaccines and improved control of infections at these sites.
Mucosal surfaces are largely protected by secretory IgA as well as transudated IgG antibodies (4). The induction of B cells in mucosal tissues following antigen exposure or infection results in the migration of B lymphocytes to mu-cosal tissues in the common mucosal immune system, thus ensuring the maintenance of secretory immunity at mucosal surfaces (1-3). T cell-mediated immunity is also a critical component of protection against mucosal pathogens. In addition to the detection of cell-mediated cytotoxicity in mucosa-associated tissues (5,6), the passive transfer of CTL is associated with the clearance of virus at mucosal surfaces and may reduce virus-related pathology (7)(8)(9)(10)(11)(12). Furthermore, as with B cells, mucosally derived T cells migrate to mucosal tissues and this homing is even more pronounced dunng re-exposure to the same pathogen (2,12).
The maintenance of T cell memory to viruses may be the result of long-lived antigen-specific lymphocytes (13)(14)(15). Alternatively, T cell memory may be maintained by constant snmulation as a result of antigen persistence (16,17), i&otypic networks (18) or cross-reactions with other antigens (19). Of interest to us, and an often ignored issue, is that the development of immune responses within the unique environments of secondary lymphoid tissues (20,21) and the homing of effector and memory lymphocytes to the tissues in which antigen exposure originally occurred (12,22,23) may restrict the maintenance and observation of T cell memory to distinct immune compartments. In-deed, in our previous studies involving mice immunized intranasally (i.n.) 1 against HSV, we observed a decline in the levels of splenic CTL such that one year after immunization we were unable to detect any anti-HSV CTL in the spleen. Interestingly, these mice were protected from an intranasal challenge with HSV-2 (24). We suspected that the lack of detectable splenic CTL at this time was a reflection of the recruitment, retention, and recirculation of specific CTL within the common mucosal immune system after intranasal immunization. In support of this theory was the concurrent observation that although systemically immunized animals displayed short-term protection from mucosal HSV-2 challenge, protection was not long-lived despite the fact that CTL specific for HSV-2 were readily detectable in the spleen (24).
Therefore, in this study we have examined the development and the long-term maintenance of CTL memory within the distinct immune compartments of the mucosal and systemic immune systems after antigen exposure in either compartment. To induce immunity to HSV, mice were immunized with a recombinant adenovlrus vector expressing the immunodominant CTL antigen of HSV, glycoprotein B (AdgB8). The CTL immune responses that developed were evaluated in the spleen and mucosal tissues of the respiratory and genital tracts.
Cytotoxic T lymphocyte memory has been functionally observed as an enhanced secondary CTL response in vitro (25)(26)(27) and in vivo (13,(28)(29)(30)(31)(32)(33) after re-exposure to antigen. We functionally examined HSV-specific memory CTL in the spleen after restimulation and expansion with antigen in vitro. CTL memory within the mucosal immune compartments of AdgB8 immunized mice was examined by detecting the rapid recall response after re-exposure to ant> gen in vivo. Our results indicate that the funcnonal presentation of short-term CTL memory is independent of the route of antigen exposure, whereas long-term CTL memory compartmentalizes over time and is dependent on the route of immunization.
Virus Strains and Inoculations. The construction of replicationcompetent recombinant adenovlrus vectors, AdgB8 and AdE3-, is reported elsewhere (33,34). In brief, AdgB8 contains the glycoprotein B (gB) gene from HSV-1 coupled to the SV40 promoter and inserted into the E3 region of human adenovlrus type 1Abbreviations used in this paper: CI, confidence interval; h.f.p., hand foot pad; HSV-2, HSV type 2; ILN, lliac lymph nodes; l.n., mtranasally; wag, intravaglnally; LU, lytac units; MLN, medlastmal lymph nodes; MOI, mulnpliclty of refection. 5. AdE3-contains a deletion in the E3 region and served as a control. The recombinant adeimviruses were grown in 293-N2S cells, purified twice on CsC1 gradients, and titered on 293 cells (35). The vaccima vector expressing gB ofHSV-1 (VacgB11) was kindly provided by B. Moss (NIH, Bethesda, MD) and its construction is reported elsewhere (36). HSV type 2 (HSV-2) strain 333 was propagated and virus tltres were determined on Vero cells. Each mouse was immunized with a total of 108 PFU of AdgB8 or AdE3-in the given volumes ofPBS pH 7.4. Mice immunized i.n. were anaesthetized with Halothane, inverted, and 10-20 }xl of virus in PBS was introduced into the nares by means ofa microplpet (24). 1.p. and hind foot pad (h.f.p.) immunizations were performed by inoculation of virus in 0.2 ml and 50 txl of PBS, respectively. Mice were challenged antravaginally (ivag) or 1.n. with 107 PFU of HSV-2 in 10 or 20 D1 of PBS, respectively. Intravaginally challenged mice were first inoculated subcutaneously with 2 mg of progestin/mouse (Depo-Provera, Upjohn, Don Mills, Ontario) 5 d before challenge, and then anesthetized using Halothane, swabbed with a cotton applicator, placed on their backs and infected for 1 h while under anesthetic.
CTL Assays. Spleen and lymph node effector cells were prepared by teasing the tissues through a stainless steel grid. Splenic CTL were examined following secondary in vitro stimulation as follows; isolated spleen cells were incubated for 6 d with gammairradiated (5,000 rad), AdgB8-infected, syngeneic MC57 cells, at an effector/stimulator ratio of 1:166 in RPMI 1640 medium with 10% FCS, 50 IxM 2-mercaptoethanol, 1% L-glutamane, penicilhn, and streptomycin. After stimulation splenic effector cells were incubated with uninfected and HSV-2-mfected (nmltiplicity of infection [1] MOI = 10, 6-h infection period) syngeneic (MC57) and allogenelc (SVBalb) targets at effector to target ratios of 80:1, 40:1, 20:1, and 10:1 in a 6-h S lCr_release assay. Inhibition of cytotoxiclty was determined by adding 100 Ixl of monoclonal antibody 145-2C11 (antl-CD3) to wells containing effector cells 1 h before addition of target cells at the 40:1 ratio. Data are expressed as percent-specific lysis = 100 X [(experimental cpm -spontaneous cpm)/(maximum release cpm -spontaneous release cpm)]. Cytotoxlc T lymphocytes from lymph nodes draining the site of infection were examined in a primary CTL assay by the protocol of Pfizenmaier et al. (37) with modifications (38). In brief, in the primary CTL assay lymphocytes from lymph nodes draining the site of infection were harvested from mice 2 or 3 d post HSV-2 challenge and incubated for 3 d (without antigen stimulation) in RPMI 1640 medium (supplemented as above). In vitro incubation (without antigen) has been shown to be necessary in the herpes system for CTL to become fully cytolync (37). After incubation, the lymph node effectors were incubated with targets in a 6 h SlCr release assay as described above. VacgB11 (MOI = 10, 16-h infection period) as well as HSV-2 infected (as above) and uninfected fibroblasts served as targets.
Determination of CTL Precursor Frequencies and Lytic Units. For determining the CTL precursor frequencies of HSV-2-spe-ClfiC splenocytes limiting dilution analysis was performed. In brief, splenocytes were isolated by Ficol gradient and txtrated into round bottom, 96-well plates (NUNC, P, oskllde, Denmark) with 12 replicates at each dilution. Feeders (irradiated splenocytes: 2,000 rids) were isolated by Flcol gradient and added at 2 × 105 mononuclear cells per well. Stimulators were irradiated (2,000 rads) with HSV-2-mfected splenocytes set at 2 × 10S/well. 5 d after incubation, the contents of each well was transferred to a corresponding well in V-bottomed NUNC plates and 5,000 51Cr labeled and HSV-2-or VacgB-infected syngenelc fibroblasts were added to each well. Plates were pulse spun up to 1,500 rpm and incubated for 6 h in a SlCr release assay. Positive wells were defined as those wells whose SlCr release exceeded the mean spontaneous release from control cultures, containing feeder cells but no responder cells, by at least three standard deviations.
The assay used to determine CTL precursor frequencies of HSV-2-specific lymphocytes from the draining lymph nodes of immunized mice was similar to that reported by Nugent et al. (31,33). This method involved expansion of CTL in the presence of exogenous cytokines but m the absence of added antigen, and results an analysis of effector CTL expanded from CTLp exclusively activated in vivo after HSV-2 challenge. Lymphocytes from lliac lymph nodes of mace infected ivag with HSV-2 were titrated into round-bottom, 96-well plates, in 100 txl of supplemented RPMI with 16 replicates for each lymphocyte concentration. Feeders (irradiated splenocytes, 2,000 rads) were isolated by Ficol gradient and added at 2 × 105 mononuclear cells per well m 100 p~l of 5 U of rlL-2 (Genzyme, Cambridge, MA), 10% (vol/ vol) Rat T-Stlm (Collaborative Biomedical Research Products, Cambridge, MA), and 50 mM co-methyl mannosade (Sigma). 5 d after incubation cultures were assessed as above for CTL precursor frequencies.
The lytlc activity of CTL in the draining lymph nodes of HSV-2-mfected tissues was estimated by determining the lytac units (LU). One LU is defined as the number of mononuclear cells required to obtain 10% specific lysls of 5,000 infected targets. The 10% level oflysis was chosen to accommodate the low levels of killing experienced by certain experimental groups. We have expressed the lytic activity of the mononuclear cells recovered from the draining lymph nodes as the number of lytic units per 107 cells. In short, mononuclear cells were harvested from the lymph nodes draining the sites of HSV-2 infection and incubated for 3 d (37) in RPMI 1640 medium with 10% fetal calf serum, 50 btM 2-mercaptoethanol, 1% L-glutamine, penicillin, and streptomycin. The lymph node effectors were then plated in twofold serial dilutions in 96-well V-bottomed plates (NUNC, P, oskllde, Denmark) with 12 rephcates at each dilution. 5,000 HSV-2-1nfected targets were added to each well and plates were briefly spun at 1,500 rpm and incubated for 6 h at 37°C in a SlCr release assay. One lytic unit was determined by estimatmg the number of lymph node effectors required to cause 10% 51Cr release by fitting a curve to the data using the equation described by Clark et al. Statistics. Frequency estimates of CTLp were calculated using X 2 analysis as described by Taswell (40), by using a computer program kindly provided by Richard Miller (University of Michigan, Ann Arbor). Estimates of CTLp frequencies were considered valid only if the plot of the logarithm of the fraction of negative cultures against the number ofresponder cells on a linear scale obeyed single-order kinetics with a probability greater than 0.05 (41).
The lytic activity for each mouse is expressed as the mean LU/ 10 v cells + SEM. Statistical analysis of LU was carried out using analysis of variance (ANOVA) based on the error associated with the line fitted to the lytic values of at least three effector to target dilutions of a given sample. Systenuc and nmcosal CTL memory in the resplratory-associated lymphoid tissue (A) and spleens (/3) of AdgB8 lmmumzed rmce Mice were lrnmunized 1 n or 1.p. and 7 mo later challenged l.n with HSV-2. The spleens and MLN were xsolated 3 d later and combined within groups The spleens (/3) were sumulated tbr 6 d with irradiated AdgB8-infected stimulators before being analyzed in a CTL assay. The MLNs (A) were cultured without antigen stimulation for 3 d before being assayed CTL acuvity is reported at E/T ratios of 40 1 (11), 20:1 (~), 10:1 ([~), and 40 1 + anti-CD3 (D).  to levels similar to that of control mice over the 19-wk period ( Fig. 1 B). Moreover, in separate experiments, splemc CTL precursor frequencies were estimated at 1 in 21,280 (95% confidence interval (CI), 12,658 to 35,714) and 1 in 500,000 (95% CI, 250,000 to 106) mononuclear cells 14 mo post i.p. or i.n. immunization, respectively. In addition, hmiting dilution analysis using fibroblasts infected with AdgB8 as stimulators gave similar results (data not shown).

R o u t e T i m e
These results indicate that while both routes of AdgB8 immunization initially resulted in the presence of anti-HSV CTL in the spleen, only i.p. immunized mice maintained long-term splemc anti-HSV memory CTL.

CTL Memory Responses in the Draining Lymph Nodes of the Respiratory Tract After AdgB8 Immunization and bmanasal HS V-2
Challenge. To investigate antigen-specific CTL memory within the respiratory tract, we examined the levels of CTL activity in the draining lymph nodes after an HSV-2 challenge. W e first determined that primary anti-HSV CTL responses in the draining lymph nodes of HSV-2-infected mucosal tissues of naive mice appeared at low levels on days 2-3 and peaked by day 5 post infection (data not shown). Therefore, in assessing memory CTL responses in the respiratory tract of AdgB8 immunized mice, mice immunized 7 mo previously with AdgB8 were challenged i.n. with HSV-2, and 3 d later the mediastinal lymph nodes (MLN) which dram the respiratory tract were examined for CTL activity. Fig. 2 A shows that only lymphocytes from the MLNs of mice immunized i.n. 7 mo previously wtth AdgB8 contained a strong anu-HSV-2 CTL recall response. In contrast, M L N cells from i.p. AdgB8 immunized mice failed to appreciably lyse HSV-2-1nfected targets (Fig.  2 A). This indicates that long-term CTL memory, as observed through a functional recall response, existed within the local mucosal i m m u n e compartment of the respiratory tract after i.n. but not i.p. AdgB8 immunization. Interest-ingly, when the spleens of these same mice were examined for antl-HSV CTL, only mice immunized i.p. demonst-rated splenocytes that recognized and killed HSV-2-infected targets (Fig. 2 B).
To further investigate the maintenance of long-term T cell memory in the respiratory tract, we examined individual mice immunized with AdgB8 and compared them to u nimmunized (naive) animals 2 d after i.n. HSV-2 challenge. with AdgB8 19 mo previously demonstrated the ability to lyse HSV-2-infected syngeneic targets. In contrast, the lymphocytes from the M L N of the naive or h.f.p, i m m unized mouse failed to appreciably lyse HSV-2-infected targets (Fig. 3). Furthermore, when the lyric activity in the MLNs of the three mice were examined, the lymphocytes in the i.n. AdgB8 immunized mouse contained significantly (P ~0.0001) and more than eight umes the lyric activity than that found in either the naive or i.p. immunized mouse (Fig. 3). These results are representative of several experiments in which individual mice were examined at late time points post immunization and at no time did we observe killing from the MLNs ofi.p, or h.f.p, immunized mice. HSV-2-specific lysis was T cell-mediated since it was completely inhibited with anti-CD3 antibody (Fig. 3). Killing was also virus-specific and MHC-restricted in the i.n. immunized mouse because uninfected and allo-infected targets were not lysed (data not shown). Figs. 2 and 3 demonstrate that long-term T cell memory responses, as observed in the draining lymph nodes, are maintained in the respiratory tract following local (i.n.) but not systemic immunization.
In Fig. 4 the presence of short-term CTL memory in the respiratory tract after i.n. or h.f.p. AdgB8 immunization was evaluated. 3 wk post immunization both these groups demonstrated a CTL recall response that was greater than  that of the naive mouse and was completely inhibitable with anti-CD3 (Fig. 4 A). As well, the lytic activity in the MLNs of i.n. or h.f.p, immunized mice was significantly greater than in the naive mouse (P ~<0.0001). In fact, there was more than eight and four times the number o f lytic units in i.n. or h.f.p, immunized mice, respectively, demonstrating that shortly after either mucosal or systemic AdgB8 immunization mice were able to mount antl-HSV-2 CTL memory responses In the mucosal-associated lymphoid tissue of the respiratory tract ( Fig. 4 A).
To determine whether the short-term CTL memory response to HSV-2 challenge was specific for gB of HSV, targets were infected with a vaccinia virus vector expressing gB of HSV (VacgB 11). The lysis of VacgB 11-infected targets was similar to that of HSV-2-infected targets (Fig. 4 B), confirming the presence of short-term CTL memory responses in both i.n. and h.f.p, immunized mice. Moreover, since the lysis and lyric activity against VacgB11 infected targets was as high as against HSV-2-infected targets, the CTL memory response to HSV-2 challenge was for the most part directed against gB o f HSV (Fig. 4 B).

CTL Memory Responses in the Draining Lymph Nodes of the Genital Tract After AdgB8 Immunization and Intravaginal
Challenge with HSV-2. W e next addressed the question of whether the maintenance of CTL memory in mucosal tissues was purely a local phenomena (i.e., due to local immunization) or was also present at distant mucosal sites.
Moreover, since HSV-2 is a sexually transmitted virus and since one o f our main interests has been the development and characterization of anti-HSV-2 immune responses in the genital tract, we investigated CTL memory responses at this site after AdgB8 immunization. Fig. 5 consists of two representative experiments showing CTL recall responses in individual mice at early (5-9 wk) and late (15-18 mo) time points post immunization (Fig. 5, A and/3). The magnitude of the primary response is indicated by the CTL activity in the naive (unimmunized) mice. In these experiments, lymphocytes were isolated from the iliac lymph nodes (ILN), which drain the genital tract, 3 d after ivag HSV-2 challenge and examined for their ability to lyse HSV-2-infected targets. Fig. 5 A demonstrates that at early time points post immunization (i.e., 5 wk) both i.n. and i.p. AdgB8 immunized mice demonstrated lysis of HSV-2-infected targets that exceeded primary responses observed in naive mice. The killing was TCR-mediated as indicated by the inhibition of lysis with anti-CD3 antibody. The lytic activity in both groups was not only similar but was significantly (P ~<0.0001) and more than 11 times greater than that observed in the naive mouse. Thus, as observed in the respiratory tract, shortly after immunization there exists a phase of CTL memory in the genital tract which is not dependent on the route of immunization. In the second experiment (Fig. 5 /3) mice were examined 9 wk post immunization and again ILN lymphocytes from the mouse immunized i.n. maintained short-term CTL memory. However, at this time point the i.p. immunized mouse lacked a significant memory response when the lysis o f targets or the lytlc activity was compared to that observed in the naive mouse (Fig. 5 B). Therefore, systemic immunization induced a phase of short-term memory in the genital tract, which we observed in the draining lymph nodes and which was absent by 9 wk post immunization.
To examine the long-term maintenance of mucosal CTL memory, mice were challenged with HSV-2 intravaginally 15 or 18 mo after AdgB8 immunization (Fig. 5, A and /3). At 18 and 15 mo, i.n. immunized mice demonstrated much higher as well as anti-CD3 inhibitable levels of killing when compared to i.p. immunized mice. Indeed, the level of lysis and the lyric activity in i.n. immunized mice at 18 or 15 mo post immunization was at least as high as that occurring at 5 or 9 wk post i.n. immunization, respectively (Fig. 5, A and B). Furthermore, in both experiments the lytic activity in ILN cells ofi.n, immunized mice was significantly (P ~<0.0001) and more than four times greater than that observed in i.p. immunized mice. In contrast, mice immunized i.p. 18 or 15 mo before lvag HSV-2 challenge lacked a memory response and their lytic activity was not significantly different from that of the naive mice (Fig. 5, A and B). These results indicate that only i.n. immunized mice maintained long-term anti-HSV CTL memory responses in the genital tract and the strength of these responses did not appear to decrease with time.  (Fig. 5 C). This is in agreement with our previous results ( Fig. 1 A) which demonstrated that i.p. AdgB8 i m m u n i z ation induced l o n g -t e r m splenic C T L memory. Interestingly, neither o f these mice demonstrated a C T L m e m o r y response in their ILNs after ivag HSV-2 challenge (Fig. 5 B).

Specificity of the CTL Memory Responses in the Draining Lymph Nodes of the Genital Tract After AdgB8 Immunization and IntravaginaI Challenge with HSV-2.
To examine the specificity and further characterize the long-term m e m o r y response m the genital tracts of i.n. i m m u n i z e d mice, the anti-HSV C T L activity in ILNs from mice immunized 5 m o previously were examined 2 d post H S V -2 challenge using targets infected with VacgB11 (Fig. 6). In addition, two systemic routes o f i m m u n i z a t i o n (i.p. and h.f.p.) were assessed and a naive mouse was included to represent the primary response to gB of HSV. Fig. 6 demonstrates that 2 d after ivag HSV-2 challenge the ILN cells in the i.n. imrnumzed mouse lysed the V a c g B l l infected targets and this lysis was completely inhibitable with a n t i -C D 3 antibody. Furthermore, the lyric activity in the 1.n. i m m u n i z e d mouse was significantly (P ~<0.0005) and more than four times greater than in the naive or systemically i m m u n i z e d mice. In contrast, the level of lysis of VacgB11 targets an naive and systemically i m m u n i z e d mice were very low and not always inhibitable with a n n -C D 3 . T h e lyric activity was also very low and n o t significantly different b e t w e e n the naive and systemically i m m u n i z e d mice (Fig. 6). These results c o nfirm that long-term immunological T cell m e m o r y is m a i ntained in the genital tracts o f mice i m m u n i z e d i.n., but not  *1 yr after AdgB8 immunization mice were challenged lvag wlth 2 X 10 v PFU of HSV-2 and 60 h later the lymphocytes from the spleens and ILN were isolated and the CTLp frequencms were determined m a limiting dilution assay. ILNs were cultured under expansion conditions without exogenous antigen. ¢Reclprocal frequency of culture wells exhibiting posinve cytolync acnvity (three standard deviations above the mean values obtained from cultures wxthout responders) were determined by mlmmal X 2 analysis. Each frequency and 95% CI represents an individual mouse (2-3 per group) and all animals were analyzed together in one experiment against VacgB11 infected targets. %Probability of obeying single-order klnencs, based upon X 2 analysis for n-1 degrees of freedom, where n was always greater than 5 for ILNs and 4 for splenocytes and represents the number of responder cell dilutions tested. biCTL_mediated lysls was determined after bulk stimulation of splenocytes as descnbed in the Materials and Methods. *2 or 12 wk after AdgB8 immunization mice were challenged lvag with 2 × 107 PFU of HSV-2 and 60 h later the lymphocytes from the spleens and ILN were isolated and the CTLp frequencies were determined In a limiting dilution assay. ILNs were cultured under expansion conditions without exogenous anngen. *Reciprocal frequency of culture wells exhihmng positive cytolytic actlvity (three standard dewatlous above the mean values obtained from cultures without responders) were deterrmned by minimal X 2 analysis. Each frequency and 95% CI represents an individual mouse (2-3 per group) and all animals were analyzed together in one experiment against VacgB 11 infected targets ~Probability of obeying single-order kinetics, based upon X 2 analysis for n-1 degrees of freedom, where n was always greater than 5 for ILNs and 4 for splenocytes and represents the number of responder cell dilutions tested.

T a b l e 1. Cytotoxic T Lymphocyte Memory Responses in the Spleen and Iliac Lymph Nodes of Mice hnmunized One Year Previously
that the recall responses in systemically immunized mice were no different than the primary responses occurring in control animals, in&caring that long-term mucosal T cell memory is maintained after mucosal but not systemic immunization. Analysis of the splenocytes of these same ammals expanded with antigen under limiting dilution conditions demonstrated that the precursor frequencies of gB-specific CTL in i.n. immunized mice were extremely low (ranging from 1 in 325543-667609). In fact, we were unable to detect any gB-specific CTL-mediated lysis after bulk stimulation of splenocytes (Table 11. In contrast, mice immunized systemically maintained relatively high frequencies of CTLp (ranging from 1 in 55068-95959) and CTL-mediated lysis was readily detectable after bulk stimulation.

Limiting Dilution Analysis of CTLp Frequencies in Mice
Shortly AfierAdgB8 Immunization. To evaluate the early development of mucosal T cell memory in AdgB8 immunized mice, we examined the CTLp frequencies in the ILNs of mice 2 or 12 wk after AdgB8 immunization. The results in Table 2 demonstrate that 60 h after an intravaginal HSV-2 challenge, the gB-specific CTLp frequencies of ILN cells in i.n. immunized mice (ranging from 1 in 2523-6845) were significantly higher than in i.p. immunized mice (at either time point; ranging from 1 in 13135-29292). Interestingly, the CTLp frequencies were similar in mace immunized i.n. at 2, 12, or 52 wk previously (ranging from 1 1886 CTL Memory in Mucosal in 2523-6845) (Tables 1 and 2). Although the gB-speclfiC CTLp frequencies in the ILNs ofi.p, immunized mice at 2 wk (ranging from 1 in 13135-15311) were not as high as in i.n. immunized mice, they were higher than at 12 wk (ranging from 1 in 17833-29292). Moreover, by 12 wk the CTLp frequencies in the ILNs ofi.p, immunized mice were similar to those observed in unimmunized mice in Table 1, suggesting that systemic immunizanon can provide mucosal T cell recall responses, however, the maintenance of this mucosal T cell memory is short-lived. The gB-specific splenic CTLp frequencies were also determined in these mice and demonstrate that both 1.n. and i.p. immunization successfully induced short-term levels of T cell memory within the systemic immune system. However, the frequencies were generally two-to threefold higher in 1.p. immunized mice. In addition, the splenic CTLp frequencies in i.p. immunized mice only slightly decreased from 2 wk (ranging from 1 in 40233-47904) to 1 yr (ranging from 1 in 55068-72671), whereas, in i.n. immunized mice there was a substantial decrease in CTLp frequencies during this period (two-to sevenfold) (Tables 1 and 2).

Discussion
T cell memory has been functionally viewed as the more rapid generation of recall responses following a second exposure to antigen (13,28,29,31). Increased numbers of antigen-specific T cell precursors as well as qualitative differences in memory T cell activation requirements likely contribute to the magnitude of recall responses (42)(43)(44)(45)(46). The ability of memory T cells to persist for long periods of time may depend on several factors, including the persistence or complexity of antigen (16,17), regulatory networks (18), cross-stimulation (19) or long-lived T cells (13)(14)(15). In addition, the maintenance of T cell memory may depend on the tissues in which exposure to antigen first occurred. Indeed, the analysis of surface markers on T cells has demonstrated that the recirculation of activated and memory lymphocytes is selective and depends on the tissues and lymph nodes from which the lymphocytes originated (47). By using recall responses we have investigated CTL memory in mucosal and systemic immune compartments after various routes of immunization with an adenovirus vector expressing gB of HSV. Our results indicate that CTL memory, when examined as a functional recall response, has both an early and a late phase which manifests in the biphasic expression of memory CTL within a given tissue depending on the site of initial antigen exposure. More specifically, the presence of systemic CTL was observed to be short-lived after i.n. immunization, however, mucosally, CTL memory was long-lived. Similarly, systemic immunization resulted in short-lived mucosal memory CTL, but long-lived systemic CTL. In addition, intranasal immunization resulted not only in long-term CTL memory in the local mucosal tissues of the respiratory tract, but also distantly in the mucosal tissues of the genital tract. This would imply that the genital tract is an implicit effector site within the common mucosal immune system. Therefore, our results demonstrate functionally the phenomenon of selective recirculation of memory lymphocytes to the tissue compartments in which sensitization to antigen first occurred. In addition, there is a short-lived period in which T cell memory can be functionally observed within the opposing immune compartment (i.e., mucosal versus systemic).
Although we have established that AdgB8 immunization results in long-lived CTL, we have not explored the underlying mechanism of this memory. However, recent evidence (48,49) suggests that infection with similar replicationcompetent recombinant adenoviruses (E3 inserts) results in only short-term expression of inserted antigen, suggesting that persistent antigen production is not the underlying mechanism responsible for the long-term maintenance of CTL memory within our system. Indeed, these studies also demonstrated that initially, vector derived antigen was present in both systemic and mucosal immune compartments regardless of the route of inoculation (48,49). This would suggest that the persistence of antigen IS unlikely driving the compartmentalized maintenance of CTL memory.
The primary observations that led to this study included the early detection of anti-HSV CTL in the spleens of i.n. or i.p. AdgB8 immunized mice, but the absence of longterm memory from the spleens of i.n. immunized mice (24). As well, mice immunized i.n. with AdgB8 were protected against heterologous intranasal challenge with HSV-2, and this protection lasted longer than in i.p. immunized mice (24). Moreover, we recently observed that i.n. AdgB8 immunization protected mice from an intravagmal HSV-2 challenge, and this protection was long-lived and significantly better than after 1.p. immunization (GaUichan, W.G., and K.L. Rosenthal, manuscript submitted for publication). By investigating these phenomena further, we show here that 1.n. and i.p. routes of AdgB8 immunization initially induced high anti-gB CTLp frequencies and following bulk stimulation, similar levels of splenic anti-HSV CTL. However, several months later, bulk stimulation of splenocytes demonstrated that mice immunized i.n. had barely detectable CTL specific for HSV. Limiting dilution analysis of mice 12-14 mo following i.n. immunization confirmed that the CTLp frequencies had decreased and had remained permanently low or undetectable. In contrast, i.p. immunization resulted in relatively high CTLp frequencies for longer than 14 mo. Others have also shown the persistence of splenic CTL following systemic exposure to gB of HSV (50). However, the transient nature of splenic CTL following 1.n. immunization may be due to the selective migration of memory T cells to mucosal sites within the common mucosal immune system (1- 3,11,12,20,23,47).
Thus, to address these seemingly conflicting observations oflong-hved mucosal protection in the absence of systemic CTL, we examined short and long-term CTL memory in mucosal tissues by assessing recall responses within mucosal associated lymph nodes after local HSV-2 challenge. At less than 9 wk after either systemic (i.p. or h.f.p.) or mucosal (i.n.) routes of AdgB8 immunization, we observed rapid recall responses in the draining lymph nodes of the lungs and genital tract. In addition, CTLp frequencies indicated that mucosal memory responses were present in animals two wk after either systemic or mucosal immunization. Thus, short-term CTL memory in mucosal tissues is not dependent on the route of immunization. In a similar manner, we examined short-term recall responses in the systemic compartment and observed that mice immunized either i.n. or systemically developed memory responses to h.f.p. HSV-2 challenge (data not shown). These results suggest that functionally there exists within a population of recently activated lymphocytes, T cells that are capable of recirculating indiscriminately throughout the body. This is interesting since recently activated lymphocytes or blasts derived from skin or mucosal tissues have been shown to preferentially but not exclusively migrate back to the tissues in which antigen exposure originally occurred (1- 3,11,12,20,23,47). Our findings may then reflect the persistence of a population of recently activated lymphocytes that are capable of responding to sites of inflammation. This is quite possible in light of work demonstrating the increased penetration of memory or activated lymphocytes into sites of antigen challenge (2,12). Alternatively, the routes of immunization used here may not reflect tissue restricted infection, resulting in some overlap of immune induction within opposing immune compartments. However, our observation of distinctively compartmentalized long-term memory responses suggests that this mechanism is unlikely to ac-count for the observed short-term phase ofT-cell memory. Moreover, it is interesting that as for short-term CTL memory, short-term protection from mucosal HSV-2 challenge (24) is not dependent on the route of immunization.
In our initial evaluation of long-term mucosal CTL memory, we examined recall responses in the respiratoryassociated lymphoid tissue and observed a dissociation of memory responses in i.n. and i.p. immunized animals. 7-19 mo after AdgB8 administration only mice immunized i.n. demonstrated CTL memory responses in the draining mediastinal lymph nodes of the respiratory tract. Interestingly, in the mice immunized 7 mo previously, only i.p. immunization resulted in the presence of long-term anti-HSV-2 CTL in the spleen. These results suggest that mucosally induced memory lymphocytes are not maintained within the spleen but perhaps within the lymphoid or extralymphoid tissues of the respiratory mucosa.
Our results, as well as our previous observations of IgA induction in the respiratory tract (24), suggest that i.n. immunization with AdgB8 stimulates the induction of immune responses in the bronchus-associated lymphoid tissue (BALT) (51). The BALT serves as part of the mucosa-associated lymphoid tissues (MALT) and as such shares organizational as well as functional similarities with other mucosal surfaces as part of the common mucosal immune system. Lymphocytes derived from one mucosal tissue can recirculate through and localize selectively within other mucosal surfaces, including the respiratory tract and uterus (1-3, 52). In agreement with this, we recently demonstrated that i.n. AdgB8 immunization results in the presence of secretory anti-HSVgB IgA in the genital tract (53) and protection from lntravagmal HSV-2 challenge (54).
To investigate the extent of mucosal CTL memory in distant mucosal tissues after i.n. AdgB8 immunization we evaluated long-term recall responses in the genital tract. Our results demonstrate that mice immunized i.n. with AdgB8 and challenged ivag up to 18 mo later with heterologous HSV-2 developed strong anti-HSV-2 CTL memory responses. Furthermore, limiting dilution analysis of the recall responses confirmed that i.n. but not systemically immunized animals maintained memory CTL for as long as one year either within the genital tract or its associated lymphoid tissues or capable of entering these sites after infection. To our knowledge, this is the first functional demonstration of long-lived CTL in local and distant mucosal tissues despite low or undetectable levels of systemic CTL.
Interestingly, the population of HSV-specific CTL that are maintained within the systemic immune system after i.p. immunization with AdgB8 are unable to mount memory responses to i.n. or ivag infection with HSV-2. This suggests that the population oflymphocytes present shortly after systemic immunization that contained T cells capable of mounting short-term mucosal memory responses were no longer present. This leaves us to suggest that dunng the early stages of an immune response there are populations of functionally active T cells that have no tissue-specific horn-ing pattern or are able to respond or enter sites of inflammation and that either do not represent the long-term population of memory T cells or phenotypically change into that population.
Our results also demonstrate that the memory responses were predominantly specific for gB of HSV since the lytic activities and the lysis of targets infected with recombinant vaccinia virus expressing gB of HSV (VacgB11) were at least as high as for HSV-2-infected targets. Moreover, analysis of CTLp frequencies specific for gB of HSV occurring in the recall response demonstrated that the memory CTL were in fact predominantly gB-specific. Cytotoxic T lymphocyte frequencies were also assessed against HSV-infected targets and the results confirmed that i.n. but not i.p. immunization induced long-term mucosal CTL memory but the absence of long-term splenic CTL memory. These results demonstrate that part of the primary CTL response to AdgB8 immunization and the recall response to HSV-2 challenge are directed towards gB of HSV. This is in contrast to recent reports by Nugent et al. (31,33) where it was shown that although a large portion of the primary response to HSV-1 inoculation involves gB-specific CTL, early secondary responses did not. Unlike in our system, the lack of an early gB-specific recall response in their model is most likely due to the fact that HSV-1 was used for both sensitization and challenge and likely reflects the interactions and complexities of numerous neutralizing anti-HSV and CTL epitopes during both periods.
Currently, many vaccines or immunotherapeutics are given systemically with the objective of providing mucosal immune functions. Our studies demonstrate that mucosal (i.n.) administration of recombinant adenovirus vectors induced long-lived antigen-specific CTL an mucosal-associated lymphoid tissues, whereas systemic administration of this vaccine induced long-lived CTL systemically but not mucosally. Development of successful vaccines against mucosal pathogens, such as HSV and HIV, will require the induction of long-lived mucosal immune responses. Recently, we showed that intranasal immunization with AdgB8 induced secretory IgA specific for gB of HSV in both the respiratory (24) and genital tracts (53). The ability of recombinant adenoviruses to induce specific mucosal humoral responses and the long-term maintenance of anti-HSV-2 CTL in respiratory and genital tissues suggest that these vectors may serve as excellent mucosal vaccines.
Our results also show that short-term mucosal CTL memory was present after both 1.n. and systemic immunization and only after several months did the memory CTL responses compartmentalize to mucosal or systemic tissues. These results have important implications with regard to the evaluation of vaccines since the time of assessment after vaccination may affect detection of CTL activity. Furthermore, the functional evaluation of memory CTL specific for mucosal pathogens should be based on assessment of CTL in mucosal-assocxated lymphoid tissues and not in the spleen.


s2fieldsofstudy:
Biology
Environmental Science

text:
Protamines of Reptiles*

We have characterized for the first time the complete primary structure of the main protamine components of the sperm from four reptiles: Chrysemys picta (turtle), Elaphe obsoleta (snake), Anolis carolinensis (lizard), and Alligator mississipiensis (crocodilian). These spe- cies were chosen to represent one of each of the main phylogenetic branches of this taxonomic group. Com- parison of these protamine sequences with those already available from other vertebrate groups allows us to define properly the chemical consensus composition of protamines and provides a unique insight into their molecular evolution and classification.

The word protamine was coined by Friederich Miescher (Miescher, 1874) to account for the nitrogenous base that he found complexed through salt-like bridges to nuclein (now DNA) from the sperm nucleus of Rhine salmon. It was not, however, until 20 years later that the true protein nature of protamines was established by Kossel working with sperm nuclei from sturgeon (Kossel, 1896a(Kossel, , 1896b. The discovery of protamines in fish was almost coincidental. Miescher's interest in the chemical composition of the cell nucleus led him to search for cells consisting mainly of nuclei. Such is the case of the sperm he obtained from salmon, which presumably was easily available at the time. Because of this historical component and also because of the easy availability of the fish system, fish protamines (particularly in salmonids) have been the most extensively characterized of all protamines to date (Dixon et al., 1985). Unfortunately, all this has inadvertently led to the use of the misleading term "typical protamines" to refer to this particular group of protamines. Indeed, protamines are widespread in other vertebrate groups (Oliva and Dixon, 1991), and they have also been found in the sperm of different groups of invertebrates (Bloch, 1969(Bloch, , 1976Subirana, 1983;. Furthermore, the primary structures of some of the invertebrate protamines strongly resemble those of the vertebrate counterparts (Daban et al., 1995).
In the case of the vertebrate protamines (see Oliva and Dixon (1991) for a comprehensive review), the mammalian protamines were the next to be widely characterized after the fish protamines. The use of polymerase chain reaction techniques has led to an outburst in the amount of sequence information within this group of vertebrates (Oliva, 1995). In recent years, a whole bounty of information has also been obtained regarding the primary structure of the protamines from monotremes and marsupials Retief et al., , 1995aRetief et al., , 1995b. In contrast to fish and mammals, there is very little information regarding the primary structure of the protamines from birds (Nakano et al., 1976;Oliva et al., 1990) and amphibians (Takamune et al., 1991;Hiyoshi et al., 1991;Ariyoshi et al., 1994;Mita et al., 1995). In the case of reptiles, the only information available to date regarding these proteins was that from PAGE 1 analysis and several amino acid compositions (Kasinsky et al., 1987;Chiva et al., 1989).
In this paper, we characterize the primary structure of the protamines from several representative species corresponding to different groups of reptiles. The major scope of this work has been to fill in the gap that presently exists regarding the primary structure of vertebrate protamines. All of this is directed toward gaining a better understanding of the chemical identity and constraints of protamines and their changes during the course of vertebrate evolution.

MATERIALS AND METHODS
Living Organisms-Crocodilians (Alligator mississipiensis, family Alligatoridae) were caught in the wild. Testis epididymis/ductus deferens were collected by Louisiana Department of Wildlife and Fisheries biologists at the Rockefeller Wildlife Refuge from nuisance/research alligators. The testes were kept in 90% ethanol (Lee et al., 1991) at 4°C, shipped to Canada by air with an appropriate CITES (Convention on International Trade in Endangered Species of Wild Fauna and Flora Export) permit, and processed for protein extraction in British Columbia, Canada. Turtles (Chrysemys picta (painted turtle) and Pseudemys scripta (pond slider), both family Emydidae), snakes (Elaphe obsoleta quadrivittata (black rat snake), family Colubridae), and lizards (Anolis carolinensis (green anole) and Sceloporus magister (spiny lizard), both family Iguanidae) either were caught in the wild in the southeastern United States or were purchased from commercial dealers throughout North America. Testis and vas deferens were collected from these organisms and either were kept in ethanol as described above or were immediately frozen and stored at Ϫ70°C until further processing. Another snake, Thamnophis scalaris (garter snake) (also family Colubridae), was captured in the wild near San Cristobal de las Casas, Chiape, Mexico. Classifications are based on Zug (1993).
Extraction and Isolation of SNBPs-Chromosomal sperm proteins were extracted and isolated as described (Ausió, 1986) with a few minor modifications. Octanol (0.2%, v/v) was added to the buffer used in the homogenization of the starting tissue (testis/vas deferens) to prevent foaming. Also, in addition to 0.2 mM phenylmethylsulfonyl fluoride, all solutions contained N ␣ -p-tosyl-L-lysine chloromethyl ketone (20 g/ml).
Enzymatic Digestion of SNBPs-Endoproteinase Glu-C (protease V8; EC 3.4.21.19; Boehringer Mannheim) digestion of the turtle protamine fractions was carried out at a 1:20 (w/w) E/S ratio for 60 min at 37°C. Chymotrypsin (EC 3.4.21.1; Boehringer Mannheim) digestion of the alligator protamine fractions was carried out at a 1:500 (w/w) E/S ratio for 60 min at room temperature. Astacus fluviatilis protease (EC 3.4.99.6; Serva) digestion of alligator and lizard protamines was performed at a 1:50 (w/w) E/S ratio for 30 min at room temperature. The protein concentration in all instances was ϳ1 mg/ml, and the buffer was 0.1 M ammonium bicarbonate, pH 8.0. Immediately after incubation for the appropriate digestion time, as established from a time course experiment, the samples were injected directly onto a reverse-phase HPLC C 18 column and eluted with different acetonitrile gradients in 0.1% trifluoroacetic acid.
Alkaline Phosphatase Treatment-Turtle (C. picta) protamine fractions were dissolved to a final concentration of ϳ0.2 mg/ml in 100 mM Tris-HCl, pH 8.0, 0.2 mM MgCl 2 , 0.2 mM ZnCl 2 . Next, calf intestinal alkaline phosphatase (EC 3.1.3.1; New England Biolabs Inc.) was added to an E/S ratio of ϳ300 units/1 mg, and the solution was incubated for 1 h at 37°C. After incubation, the sample was exhaustively dialyzed against double-distilled water and lyophilized. A protein sample treated exactly under the same conditions but in the absence of alkaline phosphatase was used as a control.
Amino Acid Analysis and Protein Sequencing-Amino acid analysis and protein sequencing were performed as described (Jutglar et al., 1991).
Sequence Analysis-Protein alignments and similarities were determined using the CLUSTAL V multiple sequence alignment program (Higgins and Sharp, 1989) and the BLITZ server at EMBL, which uses the best local similarity algorithm (Smith and Waterman, 1981). A phylogenetic tree was generated using the Phylogeny Inference package (PHYLIP) (Felsenstein, 1989).
Mass Spectrometry-Mass spectrometry analysis was carried out according to Hunt et al. (1991) as described (Carlos et al., 1993). The monoisotopic and average masses (MH ϩ ) from the protein sequences were calculated using Mac ProMass Program Version 1.05 (prepared by Dr. Terry Lee, Beckman Instruments).

RESULTS
Protamine Fractionation and Purification-The major aim of this work was the characterization of the protamines from organisms belonging to the main phylogenetic branches of reptiles (Fig. 1A). The protamine composition of several representative organisms of each such group is shown in Fig. 1B. One of the major experimental efforts in the protamine characterization was the fractionation and purification of the protein samples. The problem was compounded by the small size of the sperm samples, usually obtained from vas deferens, and also by the restricted access and seasonal availability of these organs, especially in those animals (such as alligators and snakes) that had to be collected from the wild. Also, it was not always possible to obtain organs from individuals at the same stage of gonadal maturity, and thus, the protein yields varied enormously between different individuals. On top of all this, several of the protamines from the representative reptiles studied here exhibit a large extent of protein microheterogeneity, such as in the case of the turtle C. picta and the crocodilian A. mississipiensis (Fig. 2). Although we observed that the microheterogeneity patterns were extremely conserved in all the individuals studied, the relative amounts of some of the different fractions varied slightly between individuals and also seasonally (data not shown).
Because of all this, the need to purify the fractions of interest to a high extent was deemed crucial for their sequencing analysis. The monotonous amino acid composition of some of these proteins with only a few different amino acid residues present (for example, only four in the lizard A. carolinensis) made it exceedingly difficult in these cases to use the conventional sequencing approach that consists of fractionating the protein into small pieces with proteases, followed by reconstitution of the sequence from the partial sequences of the fragments. In these instances, the proteins were thoroughly purified through multiple rounds of HPLC fractionation until extremely homogeneous fractions were obtained, as monitored by mass spectrometry. Under these circumstances, it was found possible to sequence the whole protein from beginning to end in only one sequencing run without any ambiguity, especially if the starting amount of sample analyzed was Ն2 nmol.
Major Protamine Components of the Sperm from the Turtle C. picta- Fig. 2 (A, trace Tt; and B, panel Tt) shows the extent of protein microheterogeneity of the protamines from the turtle C. picta. Despite the resolution observed in the chromatogram shown in Fig. 2A (trace Tt) and the apparent electrophoretic purity of the fractions from the corresponding peaks (Fig. 2B, panel Tt), mass spectrometry analysis indicates that each of the peaks consists of a mixture of different protein fractions (data not shown). Part of this complexity arises from the fact that some of the protamine fractions present in the different HPLC peaks shown in Fig. 2A (trace Tt) are phosphorylated (Fig. 3B). Thus, the proteins from each of the peaks recovered from the HPLC elution have to be treated with alkaline phosphatase and refractionated by HPLC (data not shown).
We noticed that although the microheterogeneity pattern (as envisaged in Fig. 3A) remained fairly constant from year to year (between different batches of turtles), the extent of phosphorylation of each of the individual fractions was very variable (data not shown). This was due presumably to differences in gonadal maturity of the different batches. To sequence the different protamine fractions, we took advantage of a unique glutamic residue in their amino acid composition. This allowed us to split the molecule into two well defined (highly homogeneous) peptides ( Fig. 3C) consisting of the N-and C-terminal domains. The peptides thus obtained were fractionated by HPLC (Fig. 3C, panel 2). After determination of their respec-tive masses (Table I) using mass spectrometry, their sequences were determined by conventional automated Edman degradation microsequencing.
The sequences of the different protamine fractions analyzed are shown in Fig. 4. The sequences of the protamine fractions from the fastest moving band (band II) on the gels (Fig. 3B) are extremely similar, with only one to three amino acid variations among all of them, thus revealing the true microheterogeneous nature of their variability. The protamine fractions running with the fast electrophoretic mobility band can be grouped into two major groups (Tt-II-1-3 and Tt-II-4 and -5), which basically differ in the hydrophobic nature of their last C-terminal amino acids. This confers a difference in the overall hydrophobicity of the molecules, which results in the high extent of resolution observed by HPLC (Fig. 3A).
We also sequenced one of the protamine components from the band of lower electrophoretic mobility (group I) (Fig. 3B). This band is only present in very low amounts (Fig. 3A), and it is in itself microheterogeneous. In fact, although we refer to the protamine fractions of this group as Tt-I-1 and Tt-I-2, each of these HPLC peaks consists of more than one fraction. The sequence of one such fraction (Tt-I-1) is shown in Fig. 4. The amino acid residues in parentheses indicate the sites of microheterogeneity. As can be seen in Fig. 4, the sequences of the protamine fractions from electrophoretic groups I and II are extremely alike except for the presence of an additional 13 amino acids in the N-terminal region of the fractions of group I (see also Fig. 9). The additional sequence is ARYRRN(RS) 3 in the case of the protamine fractions from Tt-I-1 and ARYR-RYHSHRSS in Tt-1-2 (sequence not shown). As will be discussed later, these sequence motifs are very similar to the ARYR---(BS) 3 sequence (B ϭ basic amino acid), which is commonly found in the protamines from the sperm of birds and mammals (Oliva and Dixon, 1991;Oliva, 1995).

FIG. 2.
A, reverse-phase HPLC fractionation (Vydac C 18 column (25 ϫ 0.46 cm) using 0.1% trifluoroacetic acid as eluant with an acetonitrile gradient at a flow rate of 1 ml/min). The regions of the acetonitrile gradient corresponding to the elution of histones and protamines are shown as H and P, respectively. B, electrophoretic analysis (2.5 M urea, 5% (v/v) acetic acid) of the protein fractions shown in A. The starting proteins are also shown. Tt, C. picta (turtle); Sn, E. obsoleta quadrivittata (snake); Lz, A. carolinensis (lizard); Al, A. mississipiensis (crocodilian); SL, salmine, a protamine from the salmon Oncorhynchus keta (obtained from Sigma) used as a standard. The regions of electrophoretic mobility corresponding to histones (H) and protamines (P) are indicated.
FIG. 3. A, schematic representation of the HPLC elution profile of C. picta (turtle) SNBPs (see Fig. 2A, trace Tt) to indicate the nomenclature followed to designate the different fractions sequenced (see Fig. 4). B, urea (2.5 M)/acetic acid (5%, v/v)-PAGE of some of the protamine fractions shown in A, before (Ϫ) or after (ϩ) treatment with alkaline phosphatase. C. picta protamines (lane Tt) ran in these gels in two well defined groups, I and II. Thus, fraction I-x depicts the fractions x (x ϭ 1-5) from A that ran in group I of PAGE, and fraction II-y denotes the fractions y (y ϭ 1-2) from A (shaded peaks) that ran in group II of PAGE. C, electrophoretic analysis of the peptides resulting from the digestion of different C. picta protamine fractions with protease V8 (panel 1) and elution profile (reverse-phase HPLC) of the peptides resulting from digestion of the turtle protamine fractions II-1 and II-4 with protease V8 (panel 2). The direction of electrophoresis for all the gels shown is from top (anode) to bottom (cathode).  a The nomenclature of the protein fractions is as described for Fig. 3. b The N-and C-terminal regions resulted from cleavage with staphylococcal protease V8 at a unique glutamic residue present in these proteins. c MI, monoisotopic mass; AV, average mass. Fig. 1B shows an electrophoretic analysis of the SNBP composition of two snakes (lanes 3 and 4) and two lizards (lanes 5 and 6). The electrophoretic pattern corresponding to the protamine region appears extremely conserved, not only within, but also between, each group. The different extent of histones observed in each case is due to the different degree of contamination by immature spermatogenic cells, with those showing the lower amounts of histones present (i.e. Fig. 1B, lane 3) corresponding to the less contaminated samples.

Protamines of Squamata (Lizards and Snakes)-
Reverse-phase HPLC analysis of the SNBPs and the corresponding electrophoretic analysis of the fractions from each group are shown in Fig. 2 (A, traces Sn and Lz; and B, panels Sn and Lz). As can be seen, the levels of microheterogeneity exhibited by protamines from both the snake E. obsoleta quadrivittata and the lizard A. carolinensis are much lower than in the case of the turtle C. picta (Fig. 2, A, trace Tt; and B, panel Tt).
Upon reverse-phase HPLC refractionation of each of the individual peaks shown in Fig. 2 (A, traces Sn and Lz), mass spectrometry analysis (Figs. 5B and 6B) reveals the presence of highly purified monodisperse protamine fractions. It was possible, in both instances, to obtain the complete amino acid sequence of the different fractions by direct sequencing of the molecules involved (Figs. 5A and 6A).
The average molecule masses determined from the primary structure were 5337.2 for Sn-I, 5190.0 for Sn-II, and 5068.9 for Lz, which are in excellent agreement with the masses experimentally determined by mass spectrometry. The sequences of the protamines from these organisms are very similar to the sequences of the group II protamines from the turtle C. picta (Fig. 4). There is an increase, however, in the presence of histidine and glycine as well as in the incorporation of lysine, which was absent in the turtle protamines. From the compositional point of view, the primary structure of the main protamine component from the lizard is one of the most simple protamine sequences, with only four amino acids present: arginine, glycine, lysine, and histidine.
Protamines of a Crocodilian-The apparent decrease of microheterogeneity in protamines of Squamata contrasts with the high extent of microheterogeneity found in A. mississipiensis (Fig. 2, A , trace Al; and B, panel Al). It is important to note that SNBP samples from at least 10 different alligators were analyzed in the course of this research. In each instance, the chromatographic and electrophoretic patterns observed were extremely constant, with only minor variations in the relative amounts of the different fractions, most likely resulting from the unavoidably different extent of sexual maturity. The electrophoretic pattern of the starting SNBP samples was also very similar to that reported earlier (Kasinsky et al., 1987).
One interesting feature of this system was that despite the apparently high resolutions of the HPLC fractionation ( Fig. 2A, trace Al), none of the chromatographic peaks appeared to be electrophoretically pure (Fig. 2B, panel Al). The only way to obtain pure fractions, both at the electrophoretic level (Fig. 7B) and according to mass spectrometry criteria, was to fractionate the starting SNBP extracts by ion exchange chromatography (Fig. 7A) in the presence of guanidinium chloride (Cole, 1989), followed by several rounds of reverse-phase HPLC. Thus, it looks as if the protamines from alligators consist of a complex microheterogeneous mixture of proteins. Following the aforementioned fractionation approach, we have been able to purify and sequence three of these protamine fractions (Figs. 7B and 8). The purified fractions belong to each of the three major electrophoretic bands in the starting sample (Fig. 7B, lane Al). The sequences in this case were established from the overlap of the partial sequences of HPLC-purified peptides (Fig. 7C) obtained by digestion with different proteases.
In Table II, the molecular masses of these three protamine fractions (Al-I, Al-II, and Al-III), as determined by mass spectrometry, are shown in comparison to those established from their respective amino acid sequences. One remarkable struc- FIG. 4. Amino acid sequences of the major protamine components of the sperm from the turtle C. picta. The proteins were digested with staphylococcal protease V8 (only the C-terminal peptide is underlined) upon HPLC fractionation of the resulting peptides (see Fig. 3C, panel 2). The mass of the purified peptides was determined by mass spectrometry (see Table I), and the sequences were determined using conventional Edman degradation. The nomenclature of the protamine components is as described for Fig. 3A. The asterisks designate the phosphorylated serines (see Fig. 3B) as detected by mass spectrometry. tural feature of these sequences is the presence of tryptophan in Al-I. This amino acid is seldom found in chromosomal proteins. As in the case of the protamines from Squamata, the protamine fractions from alligator are also histidine-rich. In contrast, however, lysine is not present. Instead, several tyrosine residues are present in all of them. Another important structural feature of the alligator protamines is the incorporation of the sequence ARYR---(BS) n motif (B ϭ basic amino acid) in the protamine fractions from electrophoretic bands I and II. These two electrophoretic bands can amount to 60% of the protamine components found in the sperm. Such a major presence of this motif in alligator protamines represents an important departure from the rest of the reptilian protamines, in which the ARYR---(BS) n protamine fractions were only present in very low amounts.

DISCUSSION
The Protamines of Reptiles: General Considerations-The sequence information presented in the preceding section provides a better understanding of the chemical nature of vertebrate protamines as well as of the chemical transitions undergone by these proteins during the course of vertebrate evolution. It also provides a unique insight into the general chemical composition of the protamine class of proteins. In the sections that follow, we are going to address these points specifically. Fig. 9A shows the grouping (alignment) of the protamine sequences from a representative species of each of the main phylogenetic branches of vertebrates using parsimony analysis. Several general considerations can be drawn from this sequence comparison. The first is that all vertebrate protamines seem to have a protein "core" consisting of several arginine clusters. The size of the protamine molecules has experienced a gradual but significant increase in the course of the evolution of vertebrates. This may reflect the marked physiological advantage of protamine size during the processes of displacement of the histones from the early spermatogenic stages (Oliva and Mezquita, 1986). As shown in Fig. 9A, this increase in size has occurred as a result of increasing the Nand C-terminal domains of the molecule.
Chelonian SNBPs consist of several protamine variants showing an almost identical core/C-terminal region, but differing in their N-terminal domains. This provides some indirect evidence for the possible involvement of either alternative splicing or crossing-over mechanisms leading to the acquisition of the motif ARYR(X) n (BS) 3 (where B ϭ basic residue and X is FIG. 5. A, amino acid sequences of the two major protamine components of the sperm from the snake E. obsoleta quadrivittata. Approximately 1-2 nmol of sample were loaded onto the amino acid sequencer. B, deconvoluted electrospray spectra of the two major protamine components of the sperm from the snake E. obsoleta quadrivittata. FIG. 6. A, primary structure of the major protamine component of the sperm from the lizard A. carolinensis. Approximately 1-2 nmol of protein were initially loaded onto the amino acid sequencer. B, matrix-assisted laser desorption ionization spectrum of the main protamine component of the sperm from the lizard A. carolinensis. a variable amino acid, but usually R, N, H, or C, and n ϭ 2-3), which is very conserved in the higher phylogenetic groups. The great extent of protein microheterogeneity exhibited by turtle protamines in particular and by amniote protamines in general cannot, in most instances, be accounted for by single point mutation events occurring in a precursor gene. This provides an indication of the rapid divergence of these proteins and their encoding genes (Oliva and Dixon, 1991;Oliva, 1995). Defining a Protamine: The Chemical Nature of Protamines-The availability, for the first time, of amino acid sequence information from at least one organism within each of the different vertebrate taxa allows for a better definition of SN-BPs of the protamine type . Thus, the definition proposed by Subirana (1983) (Lys ϩ Arg ϭ 45-80 mol % and Ser ϩ Thr ϭ 10 -25 mol %) can now be revised in light of this new information. With the presence in reptiles of significant large amounts of histidine and glycine as well as the presence, to a lesser extent, of lysine, we propose the following compositional definition of protamines: Arg Ն 30 mol %, His ϩ Lys ϩ Arg ϭ 45-80 mol %, and Ser ϩ Thr ϩ Gly ϭ 10 -25 mol %. This definition also takes into account the bounty of information gained in recent years on SNBPs from other invertebrate groups  that also contain protamines (Daban et al., 1995;Chiva et al., 1995).
A significant departure in this definition of the consensual protamine composition is the stress on the fact that protamines are always arginine-rich proteins regardless of their additional contents of histidine and lysine. It is important to point out that most of the lysine-rich proteins that had been previously included in this definition (Subirana, 1983) have now been shown to be related to proteins of the histone H1 family . The rest of the amino acids that appear in the amino acid composition do not seem to exhibit any major restrictions, as can be clearly seen from a glance at the sequences shown in Fig. 9A. It is also obvious from an inspection of Fig. 9A that a new amino acid, cysteine, has been incorporated into the protamine sequence during the transition from birds to mammals (Oliva, 1995), in a similar fashion to the acquisition of histidine and glycine during the transition from fish protamines to those of amphibians and reptiles. Therefore, there appears to be no reason to classify the cysteine-rich protamines from mammals within a different group of proteins. In fact, as we will discuss next, we will see that in the course of the evolution of these proteins, the appearance of amino acids other than arginine has not occurred randomly.
Sequence Alignment Analysis- Fig. 9C shows a tree obtained from sequence alignment analysis of the protamines shown in Fig. 9A. The incomplete resemblance of the tree shown in Fig.  9C to the cladogram based on the fossil record shown in Fig. 1A emphasizes the limitation of parsimony analysis (Stewart, 1993). This may arise from different causes: mainly, the reduced number of sequences available as well as their short length (30 -60 amino acids) and their arginine-rich nature (Ն30 mol %), which are the most limiting factors of this kind of analysis (Doolittle, 1986). Despite all this, the tree in Fig. 9C does provide useful information at the molecular level about the evolutionary trend of protamines in vertebrates. As in the case of the protamine P1 fraction from mammals (Oliva, 1995), the existence of a clear trend along the main phylogenetic lines can be taken as indicative of the mechanism of vertical evolution of the vertebrate protamines and their genes, rather than horizontal transmission (see Oliva and Dixon (1991)). Also, the existence of such a trend implies that in the course of evolution, the nature of the non-arginine residues that are present in protamines is not random and has been carefully selected. The analysis in Fig. 9C shows that protamines from reptiles, particularly crocodilians, are closely related to those from birds and mammals, as will be discussed next.
The ARYR Protamine Motif and the Evolution of Protamines in Amniotes-The availability, for the first time, of protamine amino acid sequences from reptiles fills an important gap in the understanding of the molecular evolution of vertebrate protamines. The presence of a common ARYR---(BS) n N-terminal sequence motif in birds, mammals, and reptiles, which is ab-  a Calculated from the Al-III sequence with His in position 10 and Arg in position 13 (see Fig. 8, Al-III).
FIG. 9. A, comparison of the sequences of protamines from vertebrates. The sequences were aligned following the method described under "Materials and Methods." The nomenclature followed for the different protamine fractions is the same as described for Figs. 2-7. The sequences for protamines other than those obtained in this work were as follows: salmine, SL-I-IV (salmon protamine from O. keta) (Hoffmann et al., 1990); protamines Bf1 and Bf2 from toad (B. japonicus) (Takamune et al., 1991); galline, Gll (fowl protamine from Gallus domesticus) (Nakano et al., 1976); protamine from a dasyurid marsupial, Sm (Sminthopsis crassicaudata) (Retief et al., 1995a); protamines P1 and P2 from boar Brp1 and Brp2 (Sus scrofa) (Tobita et al., 1983;Maier et al., 1990). B, urea/acetic acid-PAGE of the SNBPs from the following sources: SL, salmon (O. keta); Bf, toad (Bufo fowleri); Tt, turtle (C. picta); Sn, snake (E. obsoleta quadrivittata); Lz, lizard (A. carolinensis); Al, crocodilian (A. mississipiensis); Gll, rooster (Gallus gallus); Sm, dasyurid marsupial (S. crassicaudata); Br, boar (S. scrofa); and CE, chicken erythrocyte histones used as a standard. The arrowheads point to those protamine components whose sequences start with ARYR. C, Bootstrap protein parsimony tree generated from the vertebrate protamine sequences shown in A. The numbers at the nodes indicate the number of times the group consisting of the species, which are to the right of the fork, occurred among the trees out of 100 trees. The analysis was carried out using the PHYLIP package (Felsenstein, 1989). sent in fish and amphibians, indicates that the protamines in these three groups are closely related. This can be clearly seen in Fig. 9C. Thus, the incorporation of the ARYR sequence, which is usually followed by a short stretch of alternating (BS) (B ϭ basic residue and S ϭ seryl residue) (Fig. 9A), must have taken place in the transition from amphibians to the monophyletic amniote group by the Upper Carboniferous (Carroll, 1988). The acquisition of this sequence (through possible mechanisms already discussed) and/or the appearance of this protamine variant seems to have occurred gradually. Whereas in turtles, snakes, and lizards, this protein appears only as a minor component (Fig. 9B, arrowheads), in the alligator, it amounts to almost 60% of the SNBPs (Al-I and Al-II). In birds, the complete replacement by the ARYR---(BS) n -containing protein seems to have already taken place, and in mammals, this also represents the major protamine component. In the latter case, the protamine P1 component in some species coexists in variable amounts with a histidine-containing P2 component (Hecht, 1989). This protein transition affects not only the Nterminal region, but also the core region of the protamine molecule, which becomes increasingly rich in glycine (turtles, snakes, and lizards) as well as in histidine (crocodilians). In the transition to bird protamines, most of the histidines disappear. An important compositional change in this region takes place in mammals (which also affects the N-and C-terminal domains of the molecule) during the transition from Metatheria to Eutheria (Oliva, 1995) with the incorporation of cysteine. The appearance of cysteine also seems to have taken place gradually. Thus, while no cysteine can be detected in protamines from monotremes and in most marsupials (Oliva, 1995), a cysteine-containing protamine has been detected in at least one genus of shrew-like dasyurid marsupials (Planigales) (Retief et al., 1995a). This presence of cysteine in Planigales has been ascribed to a case of evolutionary convergence toward the cysteine-rich protamine P1 of eutherian mammals (Retief et al., 1995a). Yet the point mutation mechanism involved in the transition from non-cysteine-to cysteine-containing protamines in marsupials might have been similar to the ones that possibly occurred also in the transition from the non-cysteinecontaining protamine precursor to the protamines(s) found in eutherian mammals.
The different amino acid transitions observed during vertebrate protamine evolution most likely reflect the different strategies followed in the packing of chromatin and in the stabilization of the sperm nuclei. The recent acquisition of multiple cysteines (with the possibility of inter-and intramolecular bond formation) in the protamines of eutherian mammals represents an obvious advantage in this respect.
An interesting feature that comes out from the cladogram shown in Fig. 9C is that the minor histidine-containing protamine P2 that is present in some eutherian mammals appears to be more primitive than protamine P1. However, this could simply reflect the slower rate of divergence of the P2 genes when compared with P1 . As has already been pointed out earlier, it is hard to draw phylogenetic conclusions from the protein cladogram shown in Fig. 9C. Nevertheless, the significant increase in the presence of the ARYR protamine in the sperm of alligators when compared with other reptiles is fully consistent with a closer phylogenetic relation between crocodilians and birds. From looking at Fig. 1A, it remains unclear, however, what was the common selective pressure that led to the complete selection of this protamine in both birds and mammals.
Protamine Microheterogeneity-One of the striking features of protamines regarding their chemical and structural composition is their microheterogeneity (Subirana, 1983). This extent of microheterogeneity is extremely variable within and between different taxa, and hence, its origin and/or significance is still very puzzling. In salmonid fish (order Salmoniformes), most of this microheterogeneity can be accounted for by the presence of substantially polymorphic multicopy genes that encode multiple copies of protamines of restricted sequence variation (Oliva and Dixon, 1991). However, whereas in this case the protamine genes constitute a multigene family with a minimum of 15-20 members (Oliva and Dixon, 1991), in the yellow perch (order Perciformes), only one protein species seems to be present (Chao and Davies, 1992).
In amphibians, different extents of SNBP microheterogeneity have been observed in the toad Bufo japonicus (Takamune et al., 1991) and in species in the genus Xenopus (Mann et al., 1982). In birds, a copy number of only two protamine genes has been found in the two species that have been studied so far (Oliva and Dixon, 1991). In mammals, a single copy of P1 and P2 per haploid genome has been detected (Oliva and Dixon, 1991). In reptiles, as has already been pointed out, the extent of microheterogeneity also varies significantly from one group to another ( Fig. 2A), with turtle (C. picta) and alligator exhibiting the maximum extent of microheterogeneity (Figs. 1B,lane 7;and 2A,traces Tt and Al). This high extent of microheterogeneity in chelonians and crocodilians contrasts with the lower microheterogeneity observed in the phylogenetically close avian group. As noted by Subirana (1983), this broad variation in the extent of protamine microheterogeneity between different taxa points to a low physiological relevance of this phenomenon. It may, on the other hand, have significant evolutionary implications.
From a careful inspection of Fig. 9A, it appears that during the evolutionary burst leading to the appearance of amniotes, protamines underwent an important departure from the chemical and structural organization in previous vertebrate groups. While the arginine-rich nature was maintained, an important reshuffling of the non-basic residues connecting the arginine clusters as well as an increase in the overall size of the molecule occurred at this point. In this respect, turtles (Fig. 1A) represent the earliest group to undergo the transition that led to the initial reptilian protamine pattern, a pattern that already appears to be established in snakes and lizards. However, the incorporation of the ARYR---(BS) n -containing protamine as a major SNBP component did not occur until the appearance of crocodilians, and it appears to have become fully established in birds. Therefore, it looks as if the extent of microheterogeneity increases in those organisms belonging to phylogenetic groups in which the main protamine transitions have taken place. It is thus quite tempting to ascribe protamine microheterogeneity to the onset of gene duplication processes involved in the evolution of protamine genes (see Oliva and Dixon (1991)). Although the precise mechanisms involved remain to be established, the sequence data presented in this paper represent an important step in this direction. With this information, it should now be possible to design DNA primers that, in combination with the manifold molecular biology techniques presently available, should allow us to obtain the information that is ultimately required to understand the evolution of protamine genes in the vertebrates.


Deep sea microbes and DNA cloning.

RNAbor provides a new tool for researchers in the biological and related sciences to explore important aspects of RNA secondary structure and folding pathways. RNAbor computes statistics concerning d-neighbors of a given input RNA sequence and structure (the structure can, for example, be the minimum free energy (MFE) structure). A d-neighbor is a structure that differs from the input structure by exactly d base pairs, that is, it can be obtained from the input structure by adding and/or removing exactly d base pairs. For each distance d RNAbor computes the density of d-neighbors, the number of d-neighbors, and the MFE structure, or MFE structure, among all d-neighbors. RNAbor can be used to study possible folding pathways, to determine alternate low-energy structures, to predict potential nucleation sites and to explore structural neighbors of an intermediate, biologically active structure. The web server is available at http://bioinformatics.bc.edu/clotelab/

INTRODUCTION
RNA plays a surprising and previously unsuspected role in many biological processes, such as post-transcriptional regulation, conformational switches, expansion of the genetic code (such as selenocysteine insertion), ribosomal frameshift, metabolite-binding and chemical modification of specific nucleotides in the ribosome. Apart from its catalytic role as a ribonucleic enzyme (ribozyme) (1), RNA can regulate genes in several ways. For example, by hybridizing to a portion of messenger RNA, small $ 22 nt RNA molecules perform post-transcriptional gene regulation by RNA interference (RNAi), a process so important that for its discovery the 2006 Nobel Prize in Physiology or Medicine was awarded to A. Z. Fire and C. C. Mello. In addition, by very different means, RNA can perform transcriptional and translational gene regulation by allostery, where a portion of the 5 0 untranslated region (5 0 UTR) of mRNA, known as a riboswitch (2,3), undergoes a conformational change upon binding a specific ligand such as adenine, guanine or lysine.
As the field of RNomics matures, many sophisticated computational tools, e.g. RNA structure prediction, alignment and gene finding, have been developed-see (4,5) for recent overviews. Recently developed programs that are of most relevance here include the program Sfold (6,7), that computes a low energy ensemble of structures by sampling from the partition function (8), and an earlier program RNAsubopt (9) that computes all suboptimal structures within a user-specified number of kcal/mol of the minimum free energy (MFE). In addition, the program RNAshapes (10-12) provides a useful description of RNA branching structure by computing the Boltzmann probability of various shapes and also the MFE structure for various shapes. Here, an RNA shape is an equivalence class of secondary structures, describing the overall branching; for instance the shape of a typical cloverleaf tRNA would be In this article, we describe the web server RNAbor, which computes the Boltzmann probability and MFE structures which differ by base pairs from a given initial structure. Unlike most of the tools just described, which focus on the MFE structure or a low energy ensemble, RNAbor yields information concerning the secondary structure folding landscape. Potential applications of RNAbor include the design of RNA aptamers (see (13) for a suggestion how RNA might be designed to inhibit the function of the viral enzymes such as HIV-1 reverse transcriptase and hepatitis C NS3 protease), detection of conformational switches, understanding the role played by biologically active structural intermediates and improvement in secondary structure prediction.

MATERIALS AND METHODS
Let s denote a given RNA nucleotide sequence, and let S be any given secondary structure of s. The structure S could be the MFE structure of s, it could be the secondary structure obtained from the 3-dimensional X-ray conformation or by comparative sequence analysis, or it could be an arbitrary intermediate structure of particular biological significance. For an integer , a secondary structure T of s is a -neighbor of S, if S and T differ by exactly base pairs [14]. In (Freyhult, E., Moulton, V. and Clote, P. Boltzmann probability of RNA structural neighbors and riboswitch detection, submitted for publication), we describe new algorithms, which compute the number N of -neighbors, the partition function Z for -neighbors and the MFE , and the corresponding MFE structure over all -neighbors of a fixed structure S.

Computing structural neighbors
To give the reader a feeling for how the algorithms work, we present the recurrence relations to compute the number N of -neighbors of S. Let s ¼ s 1 , . . . , s n . If N i;j denotes the number of -neighbors of the substructure S ½i, j , the restriction of S to interval ½i, j of s, then the number of -neighbors of S, N ¼ N 1;n , can be computed by the following recursion: where B ¼ fAU, UA, GC, CG, GU, UGg (i.e. the set of Watson-Crick base pairs together with wobbles), b 0 ¼ 1 if j is base-paired in S ½i, j and 0 otherwise and b is the base pair distance between S ½i, j and a structure on the same interval ½i, j where a base pair between k and j has been added (taking into account all the base pairs in S ½i, j that need to be broken to allow the addition of this base pair). This approach for computing N can be extended to compute the partition function contribution, Z , of the set of -neighbors and also to compute the MFE and the MFE structure. Computations are made with respect to the Turner energy model (15,16); treatment of the dangle is similar to that in Vienna RNA Package (option -d2). The algorithms employ dynamic programming, and run in OðÁ Á n 3 Þ time and OðÁ Á n 2 Þ space, where n is the sequence length and Á is the maximum value of . Since Á can be at most n, the run time cannot be worse than Oðn 4 Þ and space no worse that Oðn 3 Þ, even if the user does not specify a value of Á. Full details of the algorithms are given in (Freyhult, E., Moulton, V. and Clote, P. Boltzmann probability of RNA structural neighbors and riboswitch detection, submitted for publication).

RESULTS
Due to the time and space constraints of the algorithm, RNA sequences may be of length up to 300 nucleotides. Sequences of length up to 60 are processed interactively and output is displayed in the user's browser window. For sequences of length 61À300, the computation is done off-line and the results are returned to the user by email; for this, the email address is required. The user can either paste an input sequence (with optional secondary structure), or upload a file of the same. The full input consists of up to four lines, illustrated by the following example.  ((((...............)))).)))).))........... 10 The temperature is set to a default value of 37 C; however the user can enter any integer temperature between 0 and 100.
The only required input is an RNA sequence s of length at most 300 nucleotides; the FASTA comment, initial secondary structure S and upper bound Á are optional inputs. If no secondary structure is given, then the initial structure S is taken to be the MFE structure, as computed by RNAfold -d2. If the optional input Á is missing, then Á is defined to equal the length n of the input sequence s; otherwise Á is the minimum of the input value and n. For each 044Á, RNAbor computes the Boltzmann probability p ¼ Z =Z, where the partition function is defined by where R is the universal gas constant and T is temperature in degrees Kelvin. Here, the summation is made over all secondary structures T of s which are -neighbors of S. The full partition function Z ¼ P Z is computed by McCaskill's algorithm (8) if Á5n.
In addition to computing probability p , RNAbor computes the number N of -neighbors of S, the MFE over all -neighbors of S and the MFE secondary structure. Tables of the values N and p , as well as their graphs, are made available as downloadable files. The five-column text file output, consisting of , p , N , MFE and the MFE structure, is depicted in Figure 1.

EXAMPLES
RNAbor can be used to generate alternative low energy structures, which differ markedly from the MFE structure, or from any initially given structure. Figure 1 shows the RNAbor output for a short 3 0 -UTR sequence of an mRNA with NCBI accession number MUSGBPS. The input structure in this example is the MFE structure (as predicted by RNAfold -d2). The RNAbor output indicates two ranges of that show higher probabilities than the rest, 0-9 and 20-24. The MFE structures at distance between 0 and 9 from the MFE structure all have very similar folds and the probability of finding the RNA in a structure at between 0 and 9 is 0.63. The probability of finding a structure at 20-24 is also relatively high, 0.35, and the MFE structures in this range are similar to each other but completely different from the MFE structure. Thus the two highly probable ranges represent two possible alternative folds of the RNA.

DISCUSSION
In this article, we have introduced the web server RNAbor, which computes the Boltzmann probability and MFE structure over all -neighbors for a given RNA sequence s and initial secondary structure S. The underlying algorithms, described in the forthcoming paper (Freyhult, E., Moulton, V. and Clote, P. Boltzmann probability of RNA structural neighbors and riboswitch detection, submitted for publication), use dynamic programming, involve the Turner energy model (15,16), and require considerable time OðÁ Á n 3 Þ and space OðÁ Á n 2 Þ resources. Figures 2  and 3 illustrate the use of RNAbor in better understanding structural aspects of a SAM riboswitch, and indicate that RNAbor should provide a useful complementary tool to programs such as Sfold and RNAshapes for analyzing the ensemble of possible secondary structures on a given RNA sequence.  The curve shows the probability, p ¼ Z =Z, for all secondary structures of RNA sequence s having base pair distance from the MFE structure S.