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Can we spike with a different enzyme to a SYBR Green Master Mix?


I followed the standard SYBR Green Protocol for doing a qPCR. For which I used

  • 10 uL of 1X SYBR Green Master Mix
  • Forward Primer and Reverse Primer (each at a final conc. = 8.5 uM)
  • Template (unknown concentration from an aptamer selection)
  • Made to 20 uL with water.

Performed the PCR amplification using

  1. 95 C Initial denaturation
  2. 95, 68, 72 - cycled 35 times.

But, one set of sample from my aptamer selection (probably high GC content) did not amplify at all. I checked on an agarose gel too.

Important Notes:

  1. My primer conc. is intentionally high - I have always amplified with such concentrations and it works perfectly using Vent Polymerase.

  2. My annealing temperature is also high at 68 C because my aptamer sequences tend to be GC Rich.

  3. Using Vent polymerase my amplification is very strong

On reading notes from vendors, I found out that the polymerase in the mastermix to be some variant of Taq Polymerase[1][3] (varies from vendor to vendor) and also it is known to poorly amplify GC Rich Sequences[2].

  1. https://www.thermofisher.com/order/catalog/product/4309155

  2. https://www.thermofisher.com/order/catalog/product/N8080241

  3. https://www.fishersci.co.uk/shop/products/powerup-sybr-green-master-mix/15366158

Hence, I wanted a suggestion from anyone working with aptamers, or if you have experience working with GC rich sequences on how I could amplify my sequences.

Much appreciated if I could have an answer before next week.

Thanks a lot for your help!!!


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In an evaluation of 24 different primer sets with PowerUp SYBR Green Master Mix, a single melt curve was obtained in 100% of reactions without the need for primer optimization or redesign. In contrast, nonspecific amplification was seen for some of the same targets in several competitors’ mixes as shown by multiple peaks in the melt curve (Figure 1). Validation of specificity in SYBR reactions is essential to data quality and validity (Bustin et al 2009). The high specificity of the PowerUp SYBR Green Master Mix allows you to spend less time and money optimizing and redesigning primers to get high-quality data.

Reproducibility is another important measure of data quality in real-time PCR reactions, and reproducibility often decays at low template concentration where the impacts of variability are exacerbated. However, PowerUp SYBR Green Master Mix, with its dual hot-start Taq DNA polymerase, demonstrates excellent reproducibility over a wide dynamic range for a variety of targets tested (Figure 2). Tighter reproducibility allows for greater significance in analyzing low-abundant transcripts and smaller fold changes.


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What is SYBR Green?

SYBR Green is a fluorescent dye used to stain nucleic acids, especially double stranded DNA in Molecular Biology. SYBR Green method is used to quantify PCR products during the real-time PCR. Once it binds with DNA, the resulting DNA-dye complex absorbs blue light and emits intense green light. It happens due to the structural change that occurs in the dye molecule upon binding with double-stranded DNA. When PCR creates more and more DNA, more dye molecules bind with DNA, generating more fluorescence. Therefore, the fluorescence increases with the PCR product accumulation. Hence, the amount of PCR product can be quantitatively measured by the SYBR Green fluorescence detection.

SYBR green dye can also be used for DNA labelling in cytometry and fluorescent microscopy. Ethidium bromide has been successfully replaced by SYBR Green since Ethidium bromide is a carcinogenic dye with disposal problems during DNA visualization in the gel electrophoresis.

There are advantages and disadvantages of SYBR green method. This method is very sensitive, inexpensive and easy to use. However, due to its ability to bind to any double-stranded DNA, nonspecific binding can lead to over quantification of PCR product.

Figure 01: SYBR Green Technique


Methods

Reagents and consumables

QIAamp® MinElute virus spin kit for DNA extraction, QIAprep® Spin Miniprep kit for plasmid extraction and QIAquick® PCR purification kit were obtained from Qiagen, Germany. TA cloning kit dual promoter (pCRII) with One Shot TOP10F`competent cells and ampicillin were obtained from Invitrogen, San Diego, California. DNA Taq polymerase, BamHI and EcoRI restriction enzymes were obtained from New England BioLabs, USA. SYBR®-Green PCR master mix, 96 well MicroAmp® fast optical reaction plates (0.1 mL capacity) and MicroAmp® optical adhesive films for real-time PCR assay were obtained from Applied Biosystems, Fostercity, CA. All the experiments were performed on StepOnePlus®-Real Time PCR Systems by Applied Biosystems, Fostercity, CA. For amplification of human torque teno virus (TTV), a set of primer pairs described previously were used (Table 3). Primers were made according to the reference strain of TTV genome TA 278 (Gen Bank acc. No. AB008394) and were synthesized by Microsynth (Switzerland) at a scale of 0.2 μmol. DNA ladders, MgCl2, dNTP’s and buffers were obtained from Fermentas Life sciences, Germany.

Samples and DNA extraction

Blood samples (5 mL) collected in EDTA tubes from 20 healthy adult volunteers and 30 randomly selected adult HSCT recipients were centrifuged at 900 g for 10 minutes to separate plasma which was immediately frozen at -20°C until used for DNA extraction. Two independent DNA extractions were performed for each of the healthy individuals along with one independent DNA extraction for HSCT recipients, each from 200 μl of plasma using QIAamp MinElute Virus Spin kit according to the manufacturer’s recommendations. DNA was eluted in 30 μL of Milli-Q water. All extracted DNA samples were stored at -20°C until the analysis. The study protocol was approved by the institution’s ethics committee and healthy donors and HSCT recipient’s samples were used after obtaining informed consent.

Construction of plasmids for standards preparation

A region of 119 bp PCR fragment of TTV genome was amplified using primers TTVf and TTVr (Table 3). Resulting amplicon was purified using QIAquick PCR Purification kit, quantified by spectrophotometer and then cloned into the TA cloning vector. The resulting plasmid was transformed into One Shot TOP10F` competent cells according to instructions provided by the manufacturer. Twelve, isolated colonies of transformed competent cells from solid luria-bertani medium containing ampicillin (100 μg/mL) were subjected for TTV insert confirmation. Each individual colony was suspended separately into 3 mL of liquid luria-bertani medium containing 100 μg/mL of ampicillin for overnight in a shaking incubator at 37°C with a speed of 225 rpm. Following overnight incubation, plasmids purification was done using QIA prep Spin Miniprep kit according to manufacturer’s instructions. Restriction enzyme digestion with EcoRI for the purified plasmids was done to confirm the presence of cloned TTV insert (119 bp) on 1.5% agarose gel electrophoresis (data not shown). TTV insert (119 bp) cloned into TA vector were sequenced for all the 12 separate clones using M-13 forward and reverse primers and confirmed by aligning with the TTV sequence (Gen Bank acc. no. AB008394). This plasmid with TTV inserts was linearized with BamHI enzyme and then used for preparation of standards in serial 10 fold dilutions from 10×10 9 copies to 20 copies/μL.

Absolute quantification of TTV DNA

PCR reaction for absolute quantification of TTV DNA using SYBR Green in a 25 μL reaction is as follows: each reaction contained 12.5 μL SYBR Green PCR master mix, 5 μL of template (serial 10 fold dilutions of the linearized plasmid standards or/ extracted DNA from the plasma samples of healthy blood donors), 1.25 μL (500 nm) of each primer (TTVF-1, TTVF-2, TTVR-1, TTVR-2) and 2.5 μL of Milli-Q water. The cycling conditions included initial activation of AmpliTaq Gold DNA polymerase (present in SYBR Green master mix) for 10 minutes at 95°C. The subsequent PCR conditions consisted of 40 cycles of denaturation at 95°C for 15 seconds and annealing and extension at 60°C for 1 minute per cycle. After real-time data acquisition, the cycle threshold value was calculated by determining the point at which the fluorescence exceeds an arbitrary threshold limit. Standards with known TTV DNA copies were prepared in two independent serial dilutions and were run in the range of 100 copies to 10×10 9 copies on four non-consecutive days to evaluate biological, inter, intraday variability and accuracy of the assay. In addition, a series of standards from one serial dilution were also run in triplicates on two different days to evaluate the intra-day and inter-day variations. The variability of the assay was evaluated by comparing the CT values run on the same day (intra-day) and on different days (inter-day) and was represented as co-efficient of variations (CV). Accuracy was calculated by taking the ratio of back calculated copies from the standard curve to the theoretical copy number of the reactions. Real-time PCR assay for test samples (HSCT recipients) and for biological replicates of each healthy individual were performed with the inclusion of TTV plasmid standards and negative controls in each run. In addition to this, precision of the assay was also checked by running known TTV positive DNA (positive controls with exact log copies/mL). The viral genomic copies per mL of plasma was calculated as described by Huang et al. [26] i.e., by multiplying the copies per reaction by a factor of 30 [30 μL extracted DNA/5 μL of template x (1 mL/200 μL plasma)].

Melting curve analysis for specificity

Following amplification, melting curve or dissociation curve analysis was performed to measure the specificity of the PCR product. The temperature program used for the melting curve analysis was 95°C for 15 seconds followed by 60°C for 1 minute and then 95°C for 15 seconds with ramp rate of +0.3°C/second.


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3. Results and discussion

To the best of our knowledge, the S-protein significantly elevated expression of Tnf-α, Il-1β, Il-6 and Mif RNAs in aged male M𠄼SF–Mϕ ( Fig.ਁ A𠄼, Suppl.  Fig.ਁ ), whereas only Tnf-α, Il-1β and Il-6 were overexpressed in young male Mϕ ( Fig.ਁ A𠄼). In contrast, IL34-Mϕ from old male mice showed an anti-inflammatory effect, with inhibition of Tnf-α and Il-6 ( Fig.ਁ G, I), while inhibition of Il-1β and Il-6 were observed in IL-34-Mϕ from young male mice ( Fig.ਁ H–I). These data indicate that the S-protein increased the expression of SASP cytokines in M𠄼SF–Mϕ while decreasing it in IL-34-Mϕ, from male mice. For young and aged female MCSF-Mϕ exposed to the S-Protein, no significant fluctuations in the expression of the Tnf-α, Il-1β, Il-6 and Mif genes were observed ( Fig.ਂ Suppl.  Fig.ਁ ). Furthermore, only a significant increase in the expression of Il-1β ( Fig.ਂ I) was detected in young female IL-34 Mϕ, whereas no changes were observed in any of the reported cytokines in aged female IL34-Mϕ. These data corresponded to a recently published observation showing that high serum levels of TNF-α and IL-6 have been identified in the COVID-19 patients, with IL-6 being significantly higher in critically ill patients [ 9 ] however, the gender or age distribution of these data have not been reported. Additionally, in COVID-19 patients, M-CSF, but not IL-34 expression, has been assessed, and found to be elevated [ 10 ]. Although, M-CSF and IL-34 have been identified in chronic inflammation, their specific contribution to the inflammatory process has not yet been detailed [ 11 ]. Our sex and age specific observations in mouse Mϕ correlated with statistical findings of high prevalence and severity of COVID-19 symptoms in senior male patients [ 2 , 12 ]. Additionally, our data clearly demonstrated the expected inflammatory phenotype induced by the SARS-CoV-2 in M-CSF, but not in IL-34, differentiated Mϕ.

The effects of SARS-CoV-2 Spike Protein on the expression of Senescence-associated secretory phenotype (SASP) markers in M𠄼SF– and IL-34-primed macrophages (Mϕ) isolated from bone marrow of young and aged male C57BL/6 miceisolated from bone marrow of young and aged male C57BL. The changes in the mRNA genes expression of pro-inflammatory cytokines (A-C, G-I), nuclear senescence-regulatory proteins (D-F, J-L) and cathepsin B, L and K (M–R), as well as the intracellular cathepsins activity (S), were assessed in M𠄼SF– or IL34-primed bone marrow derived macrophages (M𠄼SF–Mϕ, IL-34-Mϕ) after 24-h exposure to 10 ng/ml SARS-CoV-2 S-protein (n =ਅ samples/condition). Macrophages were isolated from 2-month-old and 24-month-old male and female C57BL/6 mice. Magic Red and Hoechst dyes were used to label cathepsins activity (Red) and nuclei (Blue), respectively. Scale bar: 50 μm. A one-way ANOVA with post hoc Tukey’s test for the comparisons among different groups was employed (n =ਅ samples/condition) ∗p <਀.05, ∗∗p <਀.01. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

The effects of SARS-CoV-2 Spike Protein on the expression of Senescence-associated secretory phenotype (SASP) markers in M𠄼SF– and IL-34-primed macrophages (Mϕ) isolated from bone marrow of young and aged female C57BL/6 mice. The changes in the gene expression of inflammatory cytokines (A-C, G-I), senescence markers (D-F, J-I) and cathepsins (M–R), as well as the intracellular cathepsin activity (S), were assessed in M𠄼SF– or IL-34-Mϕ after 24-h exposure to 10 ng/ml SARS-CoV-2 S-protein (n =ਅ samples/condition). Macrophages were isolated from bone marrow of 2-month-old and 24-month-old male and female C57BL/6 mice. Magic Red and Hoechst dyes were used to label cathepsins activity (Red) and nuclei (Blue), respectively. Scale bar: 50 μm. A one-way ANOVA with post hoc Tukey’s test for the comparisons among different groups was employed (n =ਅ samples/condition) ∗p <਀.05, ∗∗p <਀.01. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Since the S-protein induced production of pro-inflammatory SASP cytokines from Mϕ ( Fig.ਁ , Fig.ਂ ) and the increased susceptibility of senescent cells to viral infection [ 13 ], we next evaluated the effect of the S-protein on the expression of key nuclear senescence-regulatory proteins, including Hmgb1, p53 and p21, in MCSF- and IL34-Mϕ. Exposure to the S-protein induced the overexpression of Hmgb1, p53 and p21, by MCSF-Mϕ ( Fig.ਁ D𠄿), but not in IL34-Mϕ, isolated from old and young male mice ( Fig.ਁ J-L). In contrast, Hmgb1 and p21 were significantly overexpressed in young female IL34-Mϕ ( Fig.ਂ D,F), but not in MCSF-Mϕ. Interestingly, no changes were observed in M𠄼SF– and IL-34-Mϕ from old female mice, which may potentially be affected by the SARS-CoV-2 through different signaling pathways. HMGB1 is a damage associated molecular pattern (DAMP) alarmin, which amplifies senescence-associated inflammation [ 14 ]. Furthermore, HMGB1 is a mediator of the inflammatory cell infiltration to the lungs and contributes to the reprograming of Mϕ towards a pro-inflammatory phenotype, which is upregulated in the aging lungs and kidneys [ 15 ]. Similarly, p53 and its downstream gene p21 are upregulated in fibrotic lung disease and are known to be activated in response to DNA damage [ 16 , 17 ]. Furthermore, p53 intervenes in viral activation and has been identified in the bronchial lavage fluid of COVID-19 patients [ 18 , 19 ].

Numerous studies show cathepsins may facilitate the cellular senescence and aging-associated diseases, including osteoporosis and Alzheimer’s disease [ 20 ]. Furthermore, CatB and L are activated by the S-proteins from the SARS and MERS coronaviruses to mediate membrane fusion and subsequent release of viral RNA into the host cell [ 3 ]. Importantly, CatK is not known to be directly associated with viral infection or replication however, it does induce production of SASPs, including pro-inflammatory TNF-α, IL-6 and IL-1β cytokines, from Mϕ and Mϕ-like osteoclast precursors [ [21] , [22] , [23] , [24] ]. Thus, we finally assessed the age- and sex-dependent mRNA expression of CatB, L and K, along with their intracellular activity in M𠄼SF– and IL-34-Mϕ exposed to the S-protein using real time PCR and confocal microscopy assays, respectively.

In young males, S-protein was found to significantly elevate CatB and CatK genes expression and intracellular activities in M𠄼SF–Mϕ ( Fig.ਁ M, O), whereas CatL and CatK expressions and activities were increased in IL-34-Mϕ (Fig.ਁQ–R). Conversely, the expression and activity of CatB was inhibited while those for CatL and CatK were increased in old males M𠄼SF–Mϕ ( Fig.ਁ M–O ). No effects of S-protein were observed on IL-34-Mϕ isolated from aged mice. Our data also demonstrated that the expressions and activities of CatB and CatK were significantly increased in young female IL-34-Mϕ (Fig 2P,R) however, the expression and intracellular activities of CatB, CatL and CatK in response to S-protein were neither affected in young and old female M𠄼SF–Mϕ nor in old female IL-34-Mϕ ( Fig.ਂ ). While the importance of CatB and CatL in the SARS coronavirus-induced inflammation has been reported in previous studies [ 25 ], this study detected, for the first time, the increased expression and intracellular activity of CatK in M𠄼SF–Mϕ and IL-34-Mϕ exposed to the S-protein of SARS-CoV-2. Therefore, further investigations are warranted to elucidate the role of CatK in the age and sex associated severity of COVID-19 symptoms.


Discussion

Parr-smolt transformation in anadromous salmon is associated with a characteristic preparatory increase in gill NKA activity, which is largely completed while salmon are still in FW, allowing the smolt to move rapidly from FW to full-strength SW with minimum osmotic disturbance(Hoar, 1988). Here we provide evidence that this hypo-osmoregulatory development, as judged by changes of transcriptional, translational and activity levels of key ion-regulatory proteins, is dampened during the spring smoltification period in landlocked compared to anadromous Atlantic salmon.

Consistent with the recent findings in rainbow trout(Richards et al., 2003), we found four NKA α-isoforms (α1a, α1b, α1c and α3)to be present in salmon gills, while α2 was not detected. Present findings of a transient upregulation of gill α1b mRNA levels in anadromous salmon, concurrent with a continuous decrease of α1a, suggest that reciprocal expression of these two isoforms not only represents a mechanism through which salmonids can modulate gill NKA in response to altered salinity (Richards et al.,2003 Mackie et al.,2005 Bystriansky et al.,2006), but also constitutes an important feature underlying the preparatory increase of gill NKA activity occurring in anadromous salmon prior to SW entry. Consequently, as gill α1b mRNA is the principal isoform upregulated in anadromous smolts in the present study, showing a relative 20-fold higher upregulation than α1a from parr to smolts in May, it is likely that the preparatory increase in overall gill NKA α-subunit mRNA levels previously reported in salmon(D'Cotta et al., 2000 Seidelin et al., 2001)actually may have been a result of specific α1b isoform upregulation. In contrast to anadromous salmon, no apparent smolt-like increase of gillα1b levels occurred in landlocked salmon. On the other hand, a slight increase of α1b in juveniles landlocked during spring parallels a lower temporal increase in enzyme activity of these fish compared with anadromous smolts. Elevated NKA activity is, however, not necessarily dependent on increased α1b isoform mRNA levels. In contrast to studies on salmonids,including the present, a transient α1a upregulation was found in killifish following transfer from brackish water (BW) to SW(Scott et al., 2004a). However, α1a was also upregulated upon transfer from BW to FW, and this increase was larger and more prolonged than from BW to SW. Scott et al. further found (Scott et al.,2004b) that the differences in mortality observed between Northern and Southern killifish upon transfer from BW to FW correlated well with gill NKA activity and α1a mRNA levels. With the exception of the transient increase in SW, both studies by Scott and colleagues concur with the hypothesis of α1a having kinetic properties associated with successful ion regulation in FW (Richards et al.,2003), but the reciprocal shift between α1a and α1b isoforms seems to be specific for salmonids. Although an overall transient upregulation of gill NKA α1b mRNA, concurrent with an abrupt, sustained decrease of α1a levels in both anadromous and landlocked salmon following SW transfer, is consistent with recent findings in salmonids(Richards et al., 2003 Mackie et al., 2005 Bystriansky et al., 2006), the differences in magnitude by which these two strains respond to SW exposure illustrate an important trait associated with the development of hypo-osmoregulatory ability in salmonids the presence and magnitude of responses to salinity changes is dependent on their euryhaline capacity prior to SW entry. For instance, despite higher NKA α1b mRNA levels in anadromous salmon following SW transfer, landlocked salmon display a higher induction of α1b levels, compared with their corresponding FW values. It is therefore likely that landlocked salmon may compensate the lack of preparatory changes through higher de novo synthesis of α1b following SW transfer, as further indicated by a higher relative induction of enzyme activity among these fish in SW. Similar differences were recently observed between rainbow trout, Arctic char Salvelinus alpinus and Atlantic salmon following SW transfer(Bystriansky et al., 2006). In the case of NKA α1c, however, no apparent changes occurred in either anadromous or landlocked salmon in the present study, supporting the suggestion of a `housekeeping' function of α1c in branchial tissue of salmonids (Richards et al.,2003).

Gill Na + K + , 2Cl - cotransporter mRNA levels (A) and protein abundance (B in anadromous (closed circles) and landlocked (open circles) Atlantic salmon in FW from February 26 through June 18. Symbols for anadromous (closed triangle) and landlocked salmon (open triangle) after 96 h SW (34%thou) exposure, and anadromous (closed diamond)and landlocked salmon (open diamond) in mid-June after 1 month in SW are offset for clarity. Values are means ± s.e.m. (N=8-10). * Significant difference between strains in FW (P<0.05). Different capital and small letters denote differences (P<0.05)between timepoints within anadromous and landlocked salmon, respectively. † Significant differences between FW and SW in landlocked salmon.

Gill Na + K + , 2Cl - cotransporter mRNA levels (A) and protein abundance (B in anadromous (closed circles) and landlocked (open circles) Atlantic salmon in FW from February 26 through June 18. Symbols for anadromous (closed triangle) and landlocked salmon (open triangle) after 96 h SW (34%thou) exposure, and anadromous (closed diamond)and landlocked salmon (open diamond) in mid-June after 1 month in SW are offset for clarity. Values are means ± s.e.m. (N=8-10). * Significant difference between strains in FW (P<0.05). Different capital and small letters denote differences (P<0.05)between timepoints within anadromous and landlocked salmon, respectively. † Significant differences between FW and SW in landlocked salmon.

Gill cystic fibrosis transmembrane conductance regulator I (CFTR I) mRNA levels in anadromous (closed circles) and landlocked (open circles) Atlantic salmon in FW from February 26 through June 18. CFTR I was not measured following SW transfer. Values are means ± s.e.m. (N=8-10). * Significant difference between strains in FW (P<0.05). Different capital and small letters denote differences (P<0.05)between time points within anadromous and landlocked salmon, respectively.

Gill cystic fibrosis transmembrane conductance regulator I (CFTR I) mRNA levels in anadromous (closed circles) and landlocked (open circles) Atlantic salmon in FW from February 26 through June 18. CFTR I was not measured following SW transfer. Values are means ± s.e.m. (N=8-10). * Significant difference between strains in FW (P<0.05). Different capital and small letters denote differences (P<0.05)between time points within anadromous and landlocked salmon, respectively.

The role of the NKA α3 subunit isoform in salinity acclimation appears less important than α1 isoforms. In fact, studies in heterologous expression systems have shown that mammalian NKA isozymes show distinct affinities for Na + and K + , with the α3 isoform possessing higher Km values for Na + than the α1 and α2 NKA isozymes(Jewell and Lingrel, 1991 Blanco and Mercer, 1998 Crambert et al., 2000). Thus,a lower Na + affinity of α3 isozymes suggests that isozymes comprising this isoform may be less efficient in transporting Na + when the intracellular Na + concentration is low. However, although of a lower magnitude than α1b, a distinct transient increase of gillα3 mRNA levels in anadromous, but not landlocked salmon, suggests a significant role of this isoform during parr-smolt transformation. Consistent with findings in rainbow trout (Richards et al., 2003), neither anadromous nor landlocked salmon showed any significant increase of α3 levels following SW exposure. In tilapia, Oreochromis mossmabicus, gill α3 (twofold) and α1(fivefold) mRNA levels increase following transfer from FW to SW(Feng et al., 2002). Taken together, these results suggest that differential expression of α1 subunit isoforms is more pronounced than for α3 isoforms, probably because the α1 isoforms have kinetic properties more favorable for the differential ion transport processes of chloride cells in FW and SW(Richards et al., 2003 Evans et al., 2005). Nevertheless, further studies are necessary to ascertain the relative importance and specific roles of α1 and α3 in ion regulation.

The NKA β1-subunit is necessary for protein maturation and anchoring of the enzyme in cell membranes. Thus, co-expression of both α andβ subunits are essential for NKA function(Blanco and Mercer, 1998). The transient increase of gill NKA β1 mRNA levels in anadromous salmon in the present study is largely in accordance with previous findings(Seidelin et al., 2001). On the other hand, the lack of a preparatory increase of β1 mRNA levels in landlocked salmon, despite elevated enzyme activity in May and June, and further, decreasing levels of β1 mRNA at peak smoltification in anadromous salmon and in both strains after SW transfer, suggest additional mechanisms by which β subunits may be regulated, possibly through various osmoregulatory and/or hormone response elements(Kolla et al., 1999 Deane and Woo, 2004) or differential expression of multiple β subunit isoforms. For instance, in the European eel Anguilla anguilla, expression of gillβ 233, a duplicate copy of the NKA β1-isoform, has been found to be dependent upon the developmental stages of these fish, as upregulation only occurs in migratory silver eels, and not in adult non-migratory yellow eels following SW transfer(Cutler et al., 2000). Recently, in silico analysis of Expressed Sequence Tags has identified at least four NKA β subunit isoforms in salmonids(Gharbi et al., 2004 Gharbi et al., 2005). Assuming that multiple β subunit isoforms are present in gills, it is possible that differential expression of putative gill β1 isoforms may be similar to isoform switching of gill α1a and α1b during salmon smoltification and salinity acclimation. Alternatively, β-subunit abundance could be regulated at post-transcriptional levels, and thus differ from regulation of α-subunit synthesis.

Changes at the transcriptional level are often assumed to parallel increased protein abundance. As such, one would expect differential regulation of α-subunit isoforms at the transcriptional level to bring about differences in α protein abundance. Overall, a good correspondence between total NKA α-subunit mRNA, protein abundance and enzyme activity in the present study would suggest a coordinated regulation at the transcriptional and translational levels. This was not always the case,however, as both anadromous and landlocked salmon displayed a similar transient increase in gill α protein abundance, despite totalα-subunit mRNA levels in May being 2.5-fold higher in anadromous than landlocked salmon, based on an estimation of all four α-subunit isoforms. Similar differences in overall α-subunit mRNA and protein abundance have been found in anadromous salmonids(D'Cotta et al., 2000 Seidelin et al., 2001 Tipsmark et al., 2002),killifish (Scott et al.,2004b) and tilapia (Lee et al., 1998 Lee et al.,2003). Thus, the transient increase of α-subunit mRNA and protein abundance, with peak levels in May, concurrent with sustained elevated enzyme activity in June, indicate the importance of both transcriptional and post-transcriptional mechanisms in modulating NKA activity. Post-transcriptional mechanisms have been shown to modulate gill NKA activity in brown trout Salmo trutta(Tipsmark and Madsen, 2001),and could explain a sustained enzyme activity in June, despite a decrease in corresponding α-subunit protein and mRNA levels. The temporal switching of gill α1a and α1b mRNA between anadromous and landlocked salmon contrasts the similar transient upregulation of α-subunit protein in these two strains. Assuming that upregulation of α-isoform mRNA levels are, in fact, associated by translational changes in putative NKAα-isoform abundance, it is conceivable that the α5 antibody, which is based on conserved regions of multiple α-subunit isoforms in several vertebrate species (Takeyasu et al.,1990), may recognize all putative α-subunit isoforms, and thus account for some of the discrepancies observed in the present study. Further investigations should verify differential expression of putativeα-isoforms at the translational level in order to ascertain their physiological role in ion regulation.

While gill NKA is an essential participant in both ion secretion and uptake in gills, the basolateral NKCC and apical CFTR anion channel are considered to be primarily involved in ion secretion(Evans et al., 2005). Present findings of a preparatory transient increase of gill NKCC mRNA and protein levels in anadromous salmon are largely in accordance with previous studies in salmon (Pelis et al., 2001 Tipsmark et al., 2002). Interestingly, landlocked salmon appear to have lost the preparatory upregulation of gill NKCC mRNA associated with the parr-smolt transformation. However, like the anadromous salmon, landlocked salmon have the capacity to upregulate this transcript following SW transfer. This suggests that our TaqMan assay most likely is specific for the secretory NKCC isoform. On the other hand, two secretory isoforms, the NKCC1a and NKCC1b, have been identified in European eel, and only gill NKCC1a is upregulated following SW transfer (Cutler and Cramb,2002). Thus, one cannot exclude the possibility of more than one secretory isoform being present in salmon gills, and that these may be differentially regulated. As with NKA, there was no straightforward correspondence between NKCC mRNA and protein levels, in either anadromous or landlocked salmon, as increased NKCC protein abundance was more profound than NKCC mRNA levels, possibly reflecting a lower turnover of this protein in salmon gills. Similar differences have been observed in anadromous salmonids(Tipsmark et al., 2002) and killifish (Scott et al.,2004b). On the other hand, a distinct upregulation of NKCC protein in landlocked salmon between May and June contradicts the apparent lack of a preparatory increase at the transcriptional level in these fish. Some of the discrepancies observed in present and other studies may be ascribed to the use of the T4 antibody, as it most likely recognizes both the secretory and an absorptive isoforms (Lytle et al.,1995).

In the case of CFTR anion channel isoforms CFTR I and CFTR II, our present findings suggest that these two isoforms are differentially regulated during salmon smoltification. The continuous increase of gill CFTR I mRNA levels in anadromous salmon, and to a lesser extent in landlocked salmon, suggests a preparatory increase of this isoform during acquisition of salinity tolerance. Given that CFTR is primarily involved with ion secretion(Evans et al., 2005), it was somewhat surprising that CFTR II mRNA levels remained stable in FW among both anadromous and landlocked salmon. Assuming that both CFTR isoforms are actually inserted into the apical membrane as functional Cl - channels, it is possible that high CFTR II levels may be important for a rapid activation of CFTR when exposed to higher salinity. In fact, Singer et al.(Singer et al., 2002) found a sustained increase of gill CFTR I mRNA levels in Atlantic salmon smolts following SW, while CFTR II mRNA levels increased transiently, peaking after 24 h in SW. Further studies are clearly required to ascertain the physiological role of CFTR I and II in salmon smoltification and SW adaptation.

Salmonids display a remarkable plasticity when it comes to adjusting ion homeostasis in response to changes in environmental salinity. This plasticity may arise as part of a developmental event, or in response to salinity exposure (McCormick, 2001 Evans et al., 2005 Hiroi and McCormick, 2007). Although the landlocked salmon appears to have lost some of the developmental increase in ion transport proteins associated with preparation for SW migration, these fish seem to have retained the plasticity to respond when challenged with SW, as judged by their ability to upregulate key ion transporters and maintain low plasma Cl - levels similar to the anadromous strain. In contrast to our previous study(Nilsen et al., 2003) where this landlocked strain showed 40% mortality after 16 days in SW, no mortality occurred in the present study. One contributing factor to these contrasting observations may be the larger size of the juvenile Bleke in the present study(mean mass 39.1 g) compared with that of our previous study (mean mass 24.8 g)(Nilsen et al., 2003) when transferred to SW in May. This suggestion is in line with the general view that larger body size corresponds with greater hypo-osmoregulatory capacity in juvenile FW salmonids [see Hoar (Hoar,1988) and references therein]. However, this gradual,size-dependent increase in salinity tolerance in parr and non-anadromous species is different from the rapid and dramatic increase in salinity tolerance that develops during parr-smolt transformation [characterised by concurrent change in ontogeny, increased developmental rate and increased differentiation (McCormick and Saunders,1987)]. A threshold size for smolting in the range 9.5 cm (1+smolts) and 12 cm (2+ smolts) was described in offspring of wild broodstock(Thorpe et al., 1980),supporting our conclusions that fish from both strains were above the critical size threshold for Atlantic salmon smolt development in May (>15 cm, 39-44 g). There is further support for our view that differences between the two strains were not caused by differences in fish size: Bjerknes et al. concluded that fish size did not influence plasma osmolality or muscle water content following SW acclimation of Atlantic salmon >9.5 cm, whereas parr <9.5 cm suffered high mortalities and severe osmotic disturbance(Bjerknes et al., 1992), and Handeland and Stefansson reported higher NKA activity in small than large smolts (approx. 40 g vs 55 g), supporting smolt development being less dependent on fish size even within a wider size range than in the present experiment (Handeland and Stefannson,2001). Finally, with the exception of an increase in NKCC protein abundance, no major changes in ion regulatory parameters were observed in the juvenile Bleke between May and June, despite an increase in fish size to 54.7(±2.9) and 54.9 (±3.6) g of the Bleke and Vosso, respectively. These observations further support our view that the differences observed in May between Vosso smolts and juvenile Bleke reflect differences between strains, and are not caused by the slight (non-significant) difference in fish size in May.

The apparent loss of the preparatory osmoregulatory changes in the landlocked salmon is likely the result of natural selection, as these changes are no long necessary. Similar mechanisms have been suggested for other traits such as the loss of muscle fibers(Johnston et al., 2005), as energy may be wasted in processes that reduce their overall fitness(McDowall, 1988). In contrast,the ability to respond to SW as a protective mechanism has been retained, due to its importance in exploiting other habitats. This plasticity most likely is under less selection pressure, as the trait is only energy demanding upon SW stimulation. Even though our findings suggest the landlocked salmon do not need a preparatory increase in hypo-osmoregulatory capacity to attain ion homeostasis in SW, it must be kept in mind that these results were obtained in a protective environment with no external stressors that would otherwise be present in the wild. As stated above, the capacity to acclimate to seawater may depend on fish size, as larger fish may be able to withstand a hypersaline environment sufficiently long, allowing time for the SW-stimulated plasticity to occur, whereas the smaller fish may suffer severe salinity stress, leaving them unable to accommodate a plastic change. Support for this view was observed in a sub-experiment when fish from the present study experienced additional stress just prior to SW exposure in May. The majority of the landlocked fish died within days of SW exposure, whereas the anadromous salmon all survived (L.E., unpublished observation). Taken together, these observations indicate that stressed fish with an inadequate preparatory development of ion transporters are unable to exploit their inherent plasticity upon SW exposure.

In summary, the present study demonstrates that differential expression of gill NKA α1a, α1b and α3 isoform transcripts may, in part,be an important molecular mechanism underlying potential functional differences in NKA, both during preparatory development and during salinity adjustments in salmon. Furthermore, despite having lost some of the unique preparatory upregulation of key ion-secretory proteins associated with parr-smolt transformation, landlocked salmon have retained some hypo-osmoregulatory capacity when exposed to SW during spring.