©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The Sole Lysine Residue in Porcine Pepsin Works As a Key Residue for Catalysis and Conformational Flexibility (*)

(Received for publication, April 6, 1995; and in revised form, June 2, 1995)

Timothy J. Cottrell Linda J. Harris Takuji Tanaka Rickey Y. Yada (§)

From the Department of Food Science, University of Guelph, Guelph, Ontario, Canada N1G 2W1

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Pepsin contains a single lysine residue which protrudes from the enzyme's surface, behind the active site cleft, on the C-terminal domain. Mutations of pepsin by site-directed mutagenesis of the Lys-319 residue were generated to study the structure-function relationships. Kinetic parameters, pH activity profiles, along with conformational analysis using circular dichroism (CD), and molecular modelling were examined for the wild-type (non-mutant) and mutant enzymes. The pepsin mutations, Lys-319 Met and Lys-319 Glu, resulted in a progressive increase in the K and similar decrease in k, respectively, as well as being denatured at a lower pH than the wild-type pepsin. CD analysis indicated that mutations at Lys-319 resulted in changes in secondary structure fractions which were reflected in changes in enzymatic activity as compared to the wild-type pepsin, i.e. kinetic data and pH denaturation study. Molecular modelling of mutant enzymes indicated differences in flexibility in the flap loop region of the active site, the region around the entrance of the active site cleft, subsite regions for peptide binding, and in the subdomains of the C-terminal domain when compared to the wild-type enzyme. The results suggest that Lys-319, which is distal to the active site, is important to the flexibility/stability of the enzyme, as well as to its catalytic machinery.


INTRODUCTION

The enzymes belonging to the aspartic proteinases (EC 3.4.23.X) possess remarkable homology in their tertiary structures (Pitts et al., 1992). X-ray crystallographic data reveal that this group of enzymes shares a bilobal symmetry, with the two structural domains separated by a hinged substrate binding cleft. Two active site aspartic residues occupy the cleft to which admittance is restricted by a hinged flexible flap region (Suzuki et al., 1989). Although high in structural homology, these enzymes differ markedly in their catalytic properties, indicating that their activity is a result of subtle differences in protein structure.

Porcine pepsin (EC 3.4.23.1), a prominent member of the aspartic proteinases, was one of the first enzymes to be studied and the second to be crystallized (Northrop, 1930). Consequently, pepsin is the best understood of this family of proteinases and has been used as a model to study related enzymes (Abad-Zapatero et al., 1990; Lin et al., 1992). Pepsin is composed of 327 amino acid residues, has a calculated molecular mass of 34 kDa (Cooper et al., 1990), and is characterized by its low milk-clotting/proteolytic activity when compared with the milk-clotting enzyme of choice, chymosin (Suzuki et al., 1990). Previous investigations (Pearl and Blundell, 1984; Lin et al., 1989) into the functional activity of pepsin have been largely restricted to residues of the substrate binding pocket/active site due to their importance. This latter approach, however, assumes that the bulk of the protein structure plays a minor role in the functional activity of the enzyme, although mutations distal from the active site may influence enzymatic activity (Conrad, 1979). It has been suggested, recently, that enzymes behave more like mechanical devices than static biocatalysts implying areas external to the active site are vital to enzymatic activity (Williams, 1993).

It was the purpose of this study to observe the effect of Lys-319 mutations on the catalytic (proteolytic activity) and structural properties (changes in secondary structure, molecular modelling) of pepsin. Lys-319 is the sole lysine in the entire mature pepsin molecule. Located near the back side of the active cleft, Lys-319 is believed to have little interaction with prosegment residues during folding and is exposed to solvent in the mature enzyme (Cooper et al., 1990). It was hypothesized that the surface-protruding Lys residue plays a role in the catalysis and conformational stability/flexibility of the molecule.


EXPERIMENTAL PROCEDURES

Bacterial Strains, Plasmids, and Mutagenesis Reagents

Escherichia coli SG20252 (lon100 zba-300::Tn10) used for all protein expression along with the expression plasmid and pGBT-T19-pp containing the cDNA for porcine pepsinogen A were both kindly donated by Dr. Jordan Tang, Laboratory of Protein Studies, Oklahoma Medical Research Foundation, Oklahoma City, OK (Lin et al., 1989). The zymogen and the activated enzyme are subsequently referred to as pepsinogen and pepsin, respectively. The components used for in vitro mutagenesis were purchased from Bio-Rad (Mississauga, ON), and these included: E. coli strains, CJ236 (dut ung) and MV1190, bacteriophage M13mp19, T4 DNA polymerase, and T4 DNA ligase. Restriction endonucleases, T4 polynucleotide kinase, and calf intestinal alkaline phosphatase were purchased from Boehringer Mannheim Canada Ltd. (Laval, PQ). The two mutagenic 21-base oligonucleotide primers directing the glutamic acid, 5`-GCCAACAACGAGGTCGGCCTG-3`, and methionine, 5`-GCCAACAACATGGTCGGCCTG-3`, substitution of Lys-319 were synthesized in the Dept. of Molecular Biology and Genetics, University of Guelph. Primers used in M13 sequencing reactions were obtained from Oligo's Etc. Inc. (Wilsonview, OR).

In Vitro Mutagenesis

Site-directed mutagenesis was based on the method of Zoller and Smith(1982). Increased mutagenic efficiency was obtained using uracil-containing template (Kunkel, 1985). The procedure supplied by Bio-Rad with the in vitro mutagenesis kit was followed. The 1.3-kb (^1)fragment from the EcoRI digestion of pGBT-T19-pp, containing the pepsinogen cDNA, was subcloned into the EcoRI site of bacteriophage M13mp19. Single-stranded, uracil-containing DNA was prepared and used as a template along with the mutagenic primers for the mutagenesis reactions. Putative clones were sequenced, and mutations were confirmed in triplicate using the Core Facility for Protein/DNA Chemistry, Dept. of Biochemistry, Queen's University, Kingston, ON. The entire pepsinogen cDNA (1.3 kb) was sequenced to confirm the absence of any spontaneous mutations. The 1.3-kb EcoRI fragment from a sequenced mutant was subcloned back into pGBT-T19, and its orientation was confirmed by SstI restriction analysis. The novel proteins were expressed following transformation of E. coli SG20252 with the mutant expression plasmid. The resultant mutant proteins are referred to as Lys-319 Glu and Lys-319 Met for the glutamic acid and methionine mutations, respectively. The nonmutated protein is designated as wild-type.

Protein Purification and Refolding

E. coli SG20252, transformed with plasmid pGBT-T19-pp was cultured using a 2% inoculum in a 2-liter New Brunswick Fermentation vessel Multigen F2000 (Fisher Scientific, Mississauga, ON) containing 1.5 liters of TB medium (Sambrook et al., 1989) with 50 mg/liter ampicillin. The temperature was maintained at 37 °C, agitation was set at 200 rpm, and air was bubbled through at a rate of 4 liters/min. Fermentation was terminated when inclusion bodies were prominent (24-36 h), occupying approximately 20% of the cell volume when viewed using phase contrast microscopy. The cells were ruptured, and inclusion bodies were washed and isolated as described previously by Lin et al.(1989) with some exceptions. Following the last 0.1 M Tris-HCl, pH 10 wash, the inclusion bodies were solubilized in 50 ml of 8 M urea, 100 mM beta-mercaptoethanol at 31 °C for 2 h (Tichy et al., 1993). Residual insolubles were removed by ultracentrifugation at 286,000 g for 2 h. The clear supernatant was diluted dropwise into a slowly stirred solution of Tris-HCl, pH 8.0 (4 liters). After standing 2 h at 21 °C, the solution was cooled to 4 °C and ammonium sulfate was added to 80% saturation (Englared and Seifter, 1990). Following 12 h at 4 °C, the precipitate was collected by centrifugation at 17,000 g for 30 min, desalted by dialysis, and centrifuged at 265,000 g for 1 h to remove insoluble material. The clarified protein solution was subsequently purified by Sephacryl S-300 (Pharmacia, Uppsala, Sweden) and fast protein liquid chromatography (FPLC) with an anion exchange Mono Q HR 5/5 column (Pharmacia) as described by Lin et al.(1989).

Circular Dichroism (CD)

CD spectra of the various enzymes were measured using a Jasco J-600 spectropolarimeter (Japan Spectroscopic Co. Ltd., Tokyo, Japan) constantly flushed with nitrogen at 25 °C. Mature enzymes were prepared by incubating the zymogens at 37 °C for 20 min in buffer containing 0.1 M citrate-HCl buffer (pH 2.1). Salts and small peptides were removed by dialysis against 10 mM citrate-HCl, pH 2.1 buffer. For the pH 5.3 samples, the pH 2.1 sample was raised by adding 0.2 M sodium acetate buffer, pH 5.3, and dialyzed against 10 mM sodium acetate buffer, pH 5.3. The final protein concentration of the dialyzed samples (pH 2.1 and 5.3) was approximately 0.1 mg/ml. Samples were filtered through a 0.45-µm nylon filter and degassed under vacuum prior to analysis. Far-UV CD spectra (190-250 nm) were recorded within 30 min of sample preparation using a 0.1-cm pathlength cuvette and scanned five times. Spectra were corrected for the buffer baseline. Actual protein concentrations were determined spectrophotometrically at 280 nm using the following relationship: A of 1.0 is equal to 1.3 mg/ml (Lin et al., 1989). Secondary structure fractions for the various enzymes were determined from the far-UV CD spectra using the algorithm of Chang et al.(1978).

Kinetic Measurements

A synthetic octapeptide Lys-Pro-Ala-Glu-Phe-(p-nitro)Phe-Ala-Leu (Fusek et al. 1990) synthesized at the Central Facility of the Institute for Molecular Biology and Biotechnology, McMaster University, Hamilton ON, was used for the kinetic studies. Hydrolysis of the substrate was monitored spectrophotometrically at 300 nm in a Shimadzu UV260 recording spectrophotometer (Mandel Scientific, Guelph, ON) with external temperature control of the reaction chamber (Lauda RMT20 circulating water bath, Brinkmann Instruments Canada Limited, Rexdale, ON). The initial slopes of the progress curves were measured to yield DeltaA/min. Michaelis-Menton kinetics were observed. Plots of 1/v against 1/[S] (Lineweaver-Burk) as well as [S]/v against [S] (Hanes) permitted the fitting of a straight line by linear regression. At least five substrate concentrations from the appropriate range were used for determining initial rates (v). V(max) and K were derived from the Hanes plots due to the regularity of errors (Cornish-Bowden, 1979). Pepsinogens were activated by incubation at 37 °C for 20 min in buffer containing 0.1 M citrate and HCl (pH 2.1) at 37 °C for 20 min. The activated solutions were then placed on ice during the assays. Concentrations of active enzyme were determined by titration of a known volume of enzyme against a solution of pepstatin A (0.0833 mM in methanol). The amount of pepstatin A was taken to be equivalent to the amount of active enzyme in the sample. All kinetic studies were carried out in a 0.1 M citrate-HCl, pH 2.1 buffer except for the pH dependence experiments. Data points were based on two separate trials of duplicate determinations using substrate concentrations from 0.025 mM to 0.165 mM.

pH Activity Profiles

The pH activity profiles, over a pH range of 1.0 to 7.0, for the various enzymes were generated by incubating 0.2 nmol of the enzyme in a 0.07 mM (2 K) solution of the synthetic octapeptide Lys-Pro-Ala-Glu-Phe-(p-nitro)Phe-Ala-Leu in the appropriate buffer and monitoring the change in absorbance at 300 nm over a 5-min period at 37 °C. The following buffers were used: 0.05 M KCl-HCl for pH 1.0 through 2.0, 0.05 M glycine-HCl for pH 2.0 through 3.5, 0.05 M sodium citrate-citric acid for pH 3.5 through 5.6, and 0.05 M sodium acetate for pH 5.6 through 7. Data were reported as percent relative activity expressed as a percentage of the highest activity over the pH ranged examined for the enzyme in question. Analyses were done in duplicate.

Energy Minimization and Molecular Dynamics

Energy minimizations and molecular dynamics simulations of pepsin were performed using the program Discover 2.9.5 (Biosym Technologies, Inc., San Diego, CA) on an IBM Risc System/6000 computer. Crystal structure coordinates for wild-type pepsin were obtained from the Protein Data Bank (entry name 4PEP). Initial models of the mutant enzymes were constructed from the wild-type pepsin model by replacing the Lys-319 residue with the appropriate residue, i.e. Glu or Met. The consistent valence force field (Daube-Osguthorpe et al., 1988) was used for force field potentials throughout the calculation. In the initial minimization calculation, the steepest descents method was used, in which the line search direction is defined along the direction of the local downhill gradient -E(x, y) and was performed for 4000 iterations with a maximum derivative of 0.001 kcal/mol. The minimized models were then used for molecular dynamic simulations using the Verlet leapfrog algorithm (Verlet, 1967). The time step used was 1 fs at a temperature of 300 K, and the iteration was 5000. The neighbor list was updated every 50 time steps. After the 5-ps simulation, flexibility indices, which were the distances between appropriate alpha-carbons in the initial model and in the simulated model, were calculated at each alpha-carbon from the coordinate at every 50 fs.


RESULTS AND DISCUSSION

The amino acid sequence of porcine pepsinogen, deduced from automated dideoxy terminator sequencing confirmed the sequence as reported by Lin et al.(1989) (data not shown). The molecular mass (34 kDa) and purity of the activated product, i.e. pepsin, were confirmed using SDS-polyacrylamide gel electrophoresis and Western blotting (data not shown).

Two mutations were introduced into the porcine pepsin gene using M13 in vitro mutagenesis as described by Kunkel(1985). The efficiency of the technique (75%) was such that screening was unnecessary. Confirmation of mutagenesis was accomplished by sequencing several plaques. The confirmed mutagenic 1.3-kb EcoRI fragments from M13 were subcloned back into the original pGBT-T19 vector. Restriction analysis, using SstI, of the newly constructed expression plasmids confirmed correct construction and orientation of the insert.

Refolding of expressed proteins followed the protocol of Lin et al.(1989) and elution patterns similar to those described by these authors for the various proteins on Sephacryl S-300 gel filtration were observed (data not shown). FPLC anion exchange chromatography of the various proteins indicated that the wild-type pepsinogen eluted at approximately 15 min while Lys-319 Glu and Lys-319 Met pepsinogens eluted at approximately 16 min (data not shown). The delay in elution of the two mutant proteins was likely the result of the increase in the net negative charge of the mutants interacting with the anionite resin. The chromatographic data, however, would indicate that the refolded structures of both mutants as well as the wild-type protein were similar.

The hydrolytic and autolytic activity of pepsin is reduced at pH 6, and its activity is fully regained upon acidification (Ryle, 1966). However, at pH values above 6.0, the enzyme is rapidly and irreversibly denatured (Cooper et al., 1990). A downward shift in the upper pH limit of activity for Lys-319 Met and Lys-319 Glu, using the synthetic peptide, to just below pH 4.0 (from pH 6.0 for the native pepsin) was observed (Fig. 1) and suggests a decrease in the pH of alkaline denaturation. Further investigation into the pH activity of the mutant proteins confirmed that the inactivation pH had been shifted to approximately pH 4.0. Both mutant proteins lost activity following incubation for 4 h at pH 5.3. Alkaline denaturation of pepsin is reported to be a result of irreversible denaturation in the N terminus domain (Lin et al., 1993). Similarly, this decrease in pH activity from pH 6.0 to 4.0 for Lys-319 Met and Lys-319 Glu enzymes suggests that the mutations to Lys-319 caused parts of the C-terminal domain to be unstable similar to that observed in the N terminus by Lin et al.(1993). Although there was a substantial decrease in the inactivation pH for the mutants, there was no appreciable shift in the pH optimum (Fig. 1). Wild-type pepsin exhibited a broad range of optimum activity for this synthetic substrate, maintaining greater than 90% of its activity above pH 4.0. Both mutants displayed narrow optimum pH values around pH 2 with the Lys-319 Glu mutant displaying less than 20% activity at pH 1.4.


Figure 1: Activity profiles at varying pH using the synthetic substrate Lys-Pro-Ala-Glu-Phe-Phe(NO(2))-Ala-Leu, for wild-type pepsin (), Lys-319 Met-pepsin (bullet), and Lys-319 Glu-pepsin (). Experimental conditions are described under ``Experimental Procedures.''



The peptide, Lys-Pro-Ala-Glu-Phe-(p-nitro)Phe-Ala-Leu, was chosen as the substrate for the present study due to its high solubility characteristics and excellent sensitivity for pepsin and other aspartic proteinases (Fusek et al., 1990). The K values for cleavage of the synthetic octapeptide increased in the order of wild-type, Lys-319 Met, Lys-319 Glu pepsins (Table 1). As the K values increased, k values for the mutant enzymes decreased in the same order (wild-type pepsin, Lys-319 Met, Lys-319 Glu) suggesting that the mutations have created general conformational changes to the active site area. Furthermore, the ratio of k/K decreased in the order of wild-type pepsin, Lys-319 Met pepsin, and finally Lys-319 Glu pepsin. Taken together, these results would indicate that both the catalytic function and substrate recognition of the pepsin molecule were affected by the mutations, the Lys-319 Glu mutation having a greater effect than the Lys-319 Met mutation.



Supportive data to those observed for the kinetic parameters and pH denaturation study were seen in the analysis of CD spectral data for the various pepsins (Table 2). Comparison of secondary structure fractions of the various pepsins at pH 2.1 and 5.3 indicated changes in alpha-helix and beta-sheet fractions, the magnitude being more substantial for the mutant enzymes as compared to the wild-type enzyme. Secondary structure fractions for the wild-type pepsin were similar to those reported by previous researchers (James and Sielecki, 1986; Yada and Nakai, 1986).



Differences in catalytic parameters for the various pepsins observed at pH 2.1 (Table 1), may have been a reflection of the proportion of alpha-helix and beta-sheet fractions where the Lys-319 Glu mutant had a higher alpha-helix and lower beta-sheet content than either the wild-type or Lys-319 Met pepsins. The beta-sheet content of pepsin may be responsible for the structural flexibility (or stability) given that the CD (Yada and Nakai, 1986) and crystallographic (James and Sielecki, 1986; Abad-Zapatero et al., 1990) studies indicate that pepsin is a beta-sheet dominant protein. Examination of the beta-sheet contents from CD data indicated that the Lys-319 Glu and Lys-319 Met mutants exhibited a high degree of structural flexibility which may have resulted in decreased enzymatic activities as compared to the wild-type pepsin, i.e. the beta-sheet contents of these mutants, especially Lys-319 Glu, were much smaller than that of the wild-type as calculated from crystallographic data (approximately 40%). The substantial changes in alpha-helix and beta-sheet fractions seen in the mutant enzymes, as a function of increasing pH, corresponded to the absence of detectable enzymatic activity at pH 5.3 (see Fig. 1). However, upon examination of secondary structure fractions of the mutant proteins and the wild-type pepsin at pH 5.3, only slight differences exist indicating that the loss of enzymatic activity occurs with only minor changes in structure. Although valuable structural information is obtained from the analysis of CD data, no details regarding precise location of structural fraction(s) or identification of areas of structural change are obtained.

Recently, computer modelling/simulations have become a valuable tool in yielding information as to the possible effects of mutations on protein structure. The minimized structures of the Lys-319 Glu and Lys-319 Met pepsins were almost identical with that of wild-type pepsin except for residues around the mutated lysine residue. The total energies for the initial models of wild-type, Lys-319 Glu, and Lys-319 Met pepsins were 10,508.6 kcal/mol, 10,522.0, and 10,492.3 which decreased to 2,960.94, 2,928.81, and 2,954.63 after the energy minimizing calculations, respectively. In the minimized models, root mean square deviations of main chains were 0.008 Å between the wild-type and Lys-319 Glu mutant and 0.007 Å between the wild-type and Lys-319 Met mutant. These results suggested that the static structure of pepsin was not affected by the replacement of Lys-319; however, flexibility of the molecules may have been affected. Changes in flexibility may have accounted for the changes seen in the above CD data and the enzyme activity data of the mutant enzymes as compared to the wild-type enzyme. To estimate the flexibility of the molecules, molecular dynamics simulations on the minimized wild-type and mutant pepsin models were applied. Distances of alpha-carbon at each 50 fs from the initial position were calculated from the neighbor coordinate list yielded from the simulations. The maximum values of the alpha-carbon deviations were taken as an index of flexibility at each residue. The flexibility indices are shown in Fig. 2. Differences in the flexibility index among wild-type and mutants were plotted in order to determine the effect of the mutation of Lys-319 on the flexibility of the pepsin molecule (Fig. 3, A and B). As suggested from the CD data, substantial effects on the flexibility indices were observed in several sites which are suggested or assigned as essential segments for catalysis. These sites include a flap loop covering the active site, regions near the entrance of the active site cleft, regions which experience large movement upon inhibitor binding (subsite for peptide binding) (Chen et al., 1992), and subdomains of the C-terminal domain. In pepsin, the flap loop is a very flexible part of the enzyme. The indices of wild-type were around 1.5 to 4 Å throughout the loop. However, both mutants showed much larger indices on the loop. In the Lys-319 Met mutant, the largest index found in the molecule was observed in the loop. These differences suggest that the loops in the mutants are bending in a different manner from the loop in wild-type. Other large differences among the mutants and the wild-type were observed around the two helices in the N-terminal domain and the one loop in the C-terminal domain, all of which are at the entrance of the active cleft. In addition to these changes, larger or smaller values in the mutants than the wild-type were observed in some subsite regions for peptide binding (Chen et al., 1992). For example, Phe-117 in subsite S1 of both mutants showed a smaller index than wild-type by -1.2 Å, and Gly-34 in S1 of Lys-319 Met showed a larger index by 2.5 Å. The largest differences between the wild-type and mutant proteins, however, were observed at subdomains of the C-terminal domain. Abad-Zapatero et al.(1990) suggested that the flexibility of these subdomains plays a structural role in mediating substrate binding or determining the substrate specificity. Large differences in the indices between the mutants and wild-type were observed in the subdomain regions, i.e. from 3.0 Å at Ile-252 of Lys-319 Glu mutant to -3.7 Å at Asp-242 of Lys-319 Met mutant. These differences observed on molecular dynamics simulations suggest that the Lys-319 replacement resulted in large effects on the structural flexibility which is essential for effective catalysis.


Figure 2: The maximum movement of alpha-carbon during the molecular dynamics simulations. Each line shows the maximum movements (flexibility index) of alpha-carbons in each pepsin. Blue, red, and black are Lys-319 Glu, Lys-319 Met, and wild-type enzymes, respectively. The flap loop region, the substrate subsites, and the subdomains of the C-domain are shown with a yellow background.




Figure 3: The difference of the maximum movement at each residue during the molecular dynamics simulations. The ribbon diagram of pepsin models show the difference between wild-type and each mutant pepsin, Lys-319 Glu (A) and Lys-319 Met (B). Red portions indicate that the maximum movements (flexibility index) are larger than the counterpart of wild-type. Skyblue portions indicate the opposite. Gray portions mean both wild-type and mutant have similar indices. Thicker colored portions represent larger differences. Active Asp-32 (left) and -215 (right) are in yellow, and the Glu and Met mutants are in purple.



It has been proposed that certain enzymes are mechanical devices rather than static biocatalysts, with similarity to a pump in processing substrate (Williams, 1993). This thought gives rationality to the bulk of an enzyme's structure, which is mostly thought of as incidental when considering merely the active site and subsite binding pockets, as the catalytic machinery. Pepsin has been shown to be extremely flexible in several areas including subdomains in the C-terminal domain (Abad-Zapatero et al., 1990) and the entrance region of the active cleft (Chen et al., 1992). It is proposed that the induced mutations in pepsin at Lys-319 disrupts the enzymatic activity by changing the flexibility of the entire protein molecule and, thereby, altering the catalytic activity of the enzyme.


FOOTNOTES

*
This research was supported by a research grant from the Natural Sciences and Engineering Research Council of Canada. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence and reprint requests should be addressed. Tel.: 519-824-4120 (Ext. 8915); Fax: 519-824-0847; ryada{at}uoguelph.ca.

(^1)
The abbreviations used are: kb, kilobase(s); FPLC, fast protein liquid chromatography.


ACKNOWLEDGEMENTS

We are grateful to Drs. Xin-li Lin and Jordan Tang for supplying the pepsinogen cDNA and expression vector. We would also like to thank Massimo Marcone for his technical assistance and Tina Inalsingh for her assistance in the DNA sequencing.


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