Materials and Methods

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I. Cloning

DNA Manipulations

Preparations of lambda phage strains 9-1J and 10-1 B provided by Stephanie Moses were digested with Sal I, Bam HI, Hind III , Sal I+Hind III, Sal I+Bam HI, and Xho I (Sambrook et al., 1989). The 9-1J (860µg/µl) strain was digested with Sal I and the 10-1B (3280µg/ml) strain was digested with Bam HI for use in ligation and transformation experiments. DNA was gel purified by electroelution in a V apparatus (Medical Specialties) and was cleaned via an ethanol precipitation procedure (Sambrook et al., 1989).

Plasmid pSK- was digested with Sal I. After thirty minutes at 37C, the digested pSK was cleaned and resuspended in distilled water. The pSK was treated with calf intestinal alkaline phosphatase (CIAP, Promega) to prevent self ligation. All DNA quantities were determined by measuring absorbance of light at 260nm. Mini prep DNA was also analyzed by restriction enzyme digestions.

Ligation and Transformation

Ligation reactions were set up using 4 µl aliquots of cleaned DNA inserts. In each ligation, approximately 50pg of CIAP treated pSK was used in a 10 µl total. Reactions were incubated at 16C for thirty minutes according to the manufacturer's protocol.

50 microliters of Top 10 F´ One Shot cells and 1 µl of the ligation reactions were used in each transformation (protocol provided by Novagen). The transformed cells were spread out onto LB Ampicillin (100µg/ml), IPTG (40µl of 100mM), X-gal (32µl of 50mg/ml) plates (@ 200 µl/plate) and incubated at 37C overnight.

Mini Prep

White colonies were isolated from plates using sterile toothpicks and suspended in 2 ml of LB Ampicillin (100µg/ml). The colonies were allowed to grow overnight at 37C and 400 rpm. The plasmid DNA was isolated from the cells using an alkaline lysis procedure (Sambrook et al., 1989). The mini prep DNA was resuspended in 50 µl of TE and 5 µl of RNase A.

II. Biochemical cell mating disruption

Chloroquine treatment

NO- and cc125 cells were suspended in 400µl solutions of Nitrogen Free High Salt medium (NFHSM) and varying chloroquine concentrations (100mM, 10mM, 5mM, 1mM, or 0.5mM). All strains of cells (GA1, R3+, NO-, cc124, and cc125) were suspended in NFHSM only. Chloroquine-treated gametic cells were combined with non-chloroquine treated gametic cells and flagellar agglutination was assessed after different time intervals. Zygote formation was tested by mixing cells of the opposite mating type (chloroquine treated vs. non chloroquine treated) over night . The formation of a green pellicle indicated that zygotes had been formed.

All strains of cells (NO-, R3+, cc124, cc125, GA1, and ISO-; obtained from Duke University) were grown on 0.1mM and 0.01mM chloroquine and TAP (Tris Acetate Phosphate) plates. Cells from each plate were suspended in 400µl of NFHSM plus the appropriate concentration of chloroquine and kept under constant illumination until gametic. The mt- strains were then added to mt+ strains to determine if flagellar agglutination occurred.

Folimycin treatment

R3+, NO-, cc124, and cc125 cells were suspended in NFHSM, NFHSM plus DMSO, and NFHSM plus DMSO plus folimycin solutions. The cells were kept under lights until gametic and then agglutination between the mt+ and mt- strains was examined after different time intervals.Suspended cell numbers were quantified with a hemacytometer before mating.

III. Tetrad Analysis

Wild type cc125 cells and GA1 cells were suspended in NFHSM in Erlenmeyer flasks under constant light and at ambient temperature (Harris, 1989). Once the cells had become gametic, the two cell types were allowed to mate using the protocol outlined by Harris (1989). Zygote progeny were removed from a 1.5% bactoagar TAP plates and spread evenly onto TAP plus arginine plates. The plates were left overnight under constant illumination. Cells were resuspended in 400µl of NFHSM and put under constant light until gametic. To determine the mating types of the progeny, the cells were added separately to 10 µl of R3+ and NO- cells (selected by Dr. Goodenough for high efficiency mating).

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Results

I. Cloning

A. Sample assessment

Before the putative GA1 gene could be cloned, the six samples of lambda DNA provided by Stephanie Moses had to be analyzed in order to determine which sample had the most DNA with the least amount of RNA contamination. Stephanie had mass produced three different stocks of the 9-1 J genomic DNA (860, 540, and 560µg/µl) and 10-1 B genomic DNA (1220, 1840, 3280 µg/µl). Ten microliters of each stock sample was loaded onto a 0.77% agarose gel (Figure 6). The brightest and thickest bands in lanes 4 and 8 indicate that the 860µg/µl 9-1 J DNA and 1220µg/µl 10-1 B DNA samples contain the most DNA and thus would most likely yield the best results when trying to isolate specific fragments of the DNA for cloning. The 10-1 B genomic strains (lanes 2-4) appeared to have the most RNA contamination which can be determined by the presence of the white bands at the bottom of the gel. The smeared bands in lane 1 reveal that too much of the 1 kb molecular weight ladder was loaded in the well.

Figure 6. A 0.7% agarose gel. Ten microliters of 1 kb ladder was loaded into lane one. Ten microliters of 3280, 1840, 1220 µg/µl concentrations of 10-1 B genomic DNA were loaded into lanes 2, 3, and 4 respectively. Ten microliters of the 560, 540, and 860 µg/µl concentrations of 9-1 J genomic DNA were loaded into lanes 5, 6, and 8 respectively.



B. Restriction enzyme map verification

After determining which samples we were going to use, we needed to determine which specific fragments of the DNA we were going to target for cloning. In order to do this, we had to cut the DNA with various restriction enzymes to verify the restriction enzyme map that was provided by E. Michelle Long, a research student of Dr. Armbrust. (Figure 3; Figures 7 A and B).


Figure 7. Left Gel. A 0.7% agarose gel loaded with 9-1 J (560µg/µl) digestions. Five microliters of 1 kb ladder was loaded in lane 1. Digestions of 9-1 J with restriction enzymes Hind III, Sal I, Sal Iand Bam HI, and Sal I and Hind III were loaded into lanes 2, 3, 4, and 5 respectively. Right Gel. A 0.55% agarose gel loaded with 9-1 J(560µg/ml) and 10-1B( 1840µg/ml) DNA digestions. Five microliters of a 1kb ladder was loaded in lane 5. 9-1 J DNA digested with Bam HI was loaded into lane 1(@20µl). 10-1 B DNA digested with Xho, Sal I, and Bam HI were loaded into lanes 2, 3, and 4, respectively (@20µl each).



The following tables present the results from the digestions.


Restriction Enzymes

 Hind III Sal I Sal I and Bam HI Sal I and
Hind III		 Bam HI
Number of fragments >10 5 5 5 2
Size of fragments (kilobase, kb) corresponds to MW ladder 12, 9, 8, 4, 2 12, 9, 6.5, 3.5, 2 12, 4, 3, 2.5, 2 12, 7
Fragment totals (kb) corresponds to MW ladder 35 33 23.5 19

Table 1. Results from the 9-1 J (560µg/µl) DNA digestions with various enzymes.



Restriction Enzymes Xho Sal I Bam HI
Number of fragments 3 4 3
Size of fragments (kilobase, kb) 12, 10, 9 12, 8, 4, 3 12, 9, 7
Fragment totals (kb) 31 27 29

Table 2. Results from the 10-1 B(1840µg/ml) DNA digestions with various restriction enzymes.



Because the same size fragment of DNA is being cut in all of the digestion reactions, all of the fragments should add up to equal the same number (i.e. the molecular weight of the same piece of DNA should remain the same). The 10-1 B totals appear to be similar; however, the 9-1 J totals are very different indicating that there must be a flaw in the restriction digestion. The restriction enzyme map predicts the approximate number and sizes of fragments that should be obtained when we cut the DNA with specific enzymes (Figure 3). When restriction enzyme Hind III is used, the map predicts that two fragments, not ten, should be produced. In addition, when the DNA is cut with Sal I and Hind III, five fragments, not four, should be produced. These results indicate that the Hind III sites on the restriction enzyme map are not correct. Because the map predicts that 5 fragments should be produced when the 9-1 J DNA is cut with Sal I and because that is, in fact, what was obtained when Sal I was used, we decided to try to isolate and subclone the 8/9, 4, or 2 kb Sal I fragments with the intention of eventually ligating them together.

C. Insert isolation

Therefore, the next step was to digest a 30µl aliquot of the 9-1J with Sal I in a 50µl total. Five fragments separated from the gel as was predicted by the restriction map and the data from previous digestions (Figure 8). The 12 kb and 9kb fragments were assumed to be the arms of thelambda strain. The other three fragments (the 8/9 kb, 4kb and 2 kb fragments) were extracted from the gel and then purified.


Figure 8. A 0.55% gel of 9-1J (860µg/ml) DNA digested with Sal I. Five microliters of a 1kb ladder was loaded into lane one. The 50µl Sal I digestion was loaded into the double wide lane 2.



D. Cloning the insert

The purified DNA inserts were ligated into pSK- and transformed into Top 10 F' cells and spread onto LB ampicillin IPTG X-gal plates. Two white colonies grew on one of the 8/9 kb plates indicating that two cells had been successfully transformed by the plasmid. The DNA insert in the plasmid disrupts the production of ß-galactosidase which normally cleaves X-gal forming an insoluble blue product. Thus, cells transformed by plasmids with inserts yield white colonies that are easily distinguished from the non-transformed blue colonies on the agar. Cells from the two colonies were picked from the plate and grown overnight in 2 ml of LB ampicillin which allowed for multiple copies of the DNA insert to be harvested. Only one of the colonies that was picked successfully grew overnight. Using an alkaline lysis protocol, the DNA was collected from the cells. In order to determine if the mini prep DNA contained a fragment of interest, the mini prep DNA was digested with Sal I and without Sal I (control). The size of pSK is known to be approximately 2.9 kb (Figure 11). The digestion of the mini prep DNA should reveal a band of this size and a second band, the size of its insert. In fact, the Sal I digestion did reveal a band thatwas approximately 3.2 kb and a second band that was approximately 8/9 kb (Figure 9) . In contrast, the lane (3) loaded with just mini prep DNA revealed a single fragment of about 12/13kb. Although these results seemed to indicate that we had cloned the largest Sal I fragment, verification of our results was still necessary.

Figure 9. A 0.55% gel loaded with 8/9 MP DNA digested with and without the Sal I restriction enzyme. Five microliters of 1 kb ladder was loaded in lanes 1 and 4. The Sal I/mini prep DNA digestion was loaded in lane 2 and the no Sal I/mini prep DNA digestion was loaded in lane 3.



E. Verification of the cloned 8/9kb fragment

To verify that the insert was indeed the large 8/9 kb Sal I fragment, we had to digest the mini prep DNA with several other restriction enzymes and confirm the results with the restriction enzyme map (Figure 3). The following table shows the results obtained from the various digestions


Restriction Enzymes Sal I and Hind III Sal I and Not I Sal I and Xho Sal I and Bam HI Bam HI
Number of Fragments 10 3 4 3 2
Size of Fragments (kb) 2.5, 3.1, 7 3, 3, 3, 2.5 8.5, 3.1, 0.5 0.5, 13-14
Sum of Fragments 12.6 11.5 12.1 13.5

Table 3. Mini prep DNA digestions with various restriction enzymes.



Because the mini prep DNA digestion with Sal I and Hind III revealed 10 different bands, these results suggest, like the previous data (Table 1), that the Hind III sites must not be marked correctly on the restriction map because too many fragments are produced from this digestion than would be expected. The restriction map shows that there is one Not I site within the 8/9 kb Sal I fragment and, therefore, when the mini prep DNA is digested with both Sal I and Not I, the 8/9 kb fragment would but cut into two smaller fragments (@2 and 7kb). Our results confirmed this prediction (Figure 10). In addition, the restriction map shows that there are two Xho I sites within the 8/9 kb Sal I fragment and predicts that when the mini prep DNA is cut with both Sal I and Xho I two 3 kb fragments and one 2 kb fragment would be produced. Again, our results confirmed this prediction (Figure 10).




The DNA restriction map also shows that there is one Bam HI site located in the 8/9 kb Sal I fragment and suggests that when the mini prep DNA is cut with both Bam HI and Sal I, the digestions should produce a 0.5 kb fragment and a 7.5-8.5 kb fragment. As can be seen in Figure 12 A, our results confirm this prediction. Once we had verified that we had cloned the 8/9 kb Sal I fragment, we needed to determine the orientation of the 8/9 insert within pSK-. Therefore, we did an additional digestion using only Bam HI and the mini prep DNA. We know that the plasmid is approximately 2.9 kb and that it has a Bam HI site approximately 45 bp outside of the Sal insert site. A digestion of the mini prep DNA with only Bam HI should reveal a small band (@0.5kb) and a larger band (@13kb) or two larger bands (@7/8kb), depending on how the insert is oriented in the plasmid (Figure 12 B). The digestion with Bam HI revealed a 13-14 kb band and a 0.5 kb band. These results indicate that the orientation in Figure 11 A is the orientation of the 8/9 kb fragment within pSK-.




The next goal in the cloning process was to isolate the second half of the gene and successfully ligate that fragment into pSK- and transform a cell. The fragment of interest is a 7kbBam HI fragment. If this fragment can be successfully subcloned, then the two cloned fragments could be ligated together and transformed into GA1 cells. If the phenotype is complemented then the entire putative GA1 gene will have been cloned. Recently, some of the DNA was digested with Bam HI and a 12 kb, 9 kb and 7 kb fragment was separated by the gel (Figure 13). The 7 and 9kb fragments were removed from the gel and purified. The concentration of the purified DNA is 120µg/ml. This DNA stock was sent to Dr. Armbrust in Washington to continue our efforts in subcloning the 7 kb Bam HI fragment.




III. Biochemical Disruption of Cell Mating

The sequence data of the cDNA that Dr. Armbrust had obtained from wild type GA1 cDNA was translated into an amino acid sequence and sent via email to Genbank at the National Institutes of Health where the sequence was compared to all amino acid sequences that are stored in the data bank. The top hits indicated that our sequence was very similar to a gene encoding an H+-ATPase (Table 4). Having this information, a model was proposed as the mechanism causing the mating disruption in GA1 cells (Figure 14). In addition, the accession numbers of the top ten hits weresent via email to the "retrieve server" at Genbank which provided the amino acid sequences and sources of the genes. Using the MACDNAsis program, the amino acid sequences were multiplyaligned with the amino acid sequence of the GA1 cDNA. There was a 31% sequence similarity between the GA1 sequence and the Leishmania donovania sequences and an 18% sequence similarity to the Heterosigma akashiwo sequence (Appendix A). Leishmania donovania and Heterosigma akashiwo are both unicellular, flagellated organisms .



We wanted to see if the chloroquine had the same effects on two other strains of cells, cc124 and cc125, that are more like GA1 cells. The R3+ and NO- cells were specifically engineered to have a higher mating efficency. Therefore, the second chloroquine experiment used cc124 and cc125 cells. cc124 cells were suspended in a 1mM, 0.1 mM, and 0.01 chloroquine plus NFHSM solutions because they, like GA1, are mt- cells. The cc124 and cc125 cells take longer to become gametic than the R3+ and NO- cells, so mating was observed after 6 and 7 hours. When the cc124 (1mM) was combined with cc125, some agglutination and cell wall loss was observed. In comparison to the positive control (untreated cc124 X cc125), the amount of agglutinating cells was significantly less. The zygote test revealed a green pellicle for all of the matings; however, the pellicle formed from the cc124 (0.1 and 0.01mM) matings with the cc125 was more weak as compared to the others.



The previous two experiments demonstrated that it takes a relatively long amount of time for chloroquine to show any effects on all strains of cells. In order to see if the cells could survive at all and to see what effects long term chloroquine treatment would have on vegetative cell-mating ability, we decided to actually grow cells on agar plates that contained chloroquine. NO-, R3+, cc124, cc125, GA1, and ISO- cells were grown on 0.1 mM and 0.01 mM chloroquine and TAP plates. Only R3+ cells, which appeared to be very dry, grew on the 0.1mM chloroquine plates. All cell types grew on the 0.01 mM chloroquine plates and the cells did not look dry. Cells from all of the plates were made gametic in the appropriate chloroquine plus NFHSM solution. Different cells were combined and then viewed under a microscope to determine if agglutination was occurring. The cc124 and cc125 grown on the 0.01 mM chloroquine plates did not appear to mate with any of the cell strains (Table 7). The R3+ and N0- cells, however, did appear to mate normally with most strains. Although we observed that the chloroquine was having some type of effect on the cells and through personal communication we knew that chloroquine was only 60-70% effective in inhibiting lysosomal activity in lymphpcytes (Mariolinsa Salio), we were unable to mimic the GA1 mutant phenotype and, therefore, decided to abandon the chloroquine experiments.

cc124

NO-

cc125

R3+

mating

zygote test

-

-

yes

green pellicle

+

+

yes

green pellicle

+

+

no

green pellicle

-

+ *

yes

ND

+

-

no

ND

+

+

no

ND

+

+

no

ND

+

+

no

ND

B. Folimycin Treatment

Because we were unsuccessful in trying to raise the pH of the intracellular compartments using chloroquine, we decided to us an H+-ATPase inhibitor in normal cells. By using an H+- ATPase inhibitor, the vacuolar pH within the cell would be raised perhaps causing a disruption in the glycosylation process. Therefore, our next experiment involved the use of the H+-ATPase inhibitor, folimycin (Marshanky and Vinay, 1996; Muroi et al. 1993). Folimycin was dissolved in DMSO; therefore, control solutions containing only NFHSM, and DMSO plus NFHSM were made in addition to the experimental solution of NFHSM plus DMSO plus folimycin. The DMSO appeared to have no effect on the cells as they all mated successfully after becoming gametic in the NFHSM and DMSO solution (Table 8). R3+ and NO- cells did not appear to be affected by thefolimycin as the cells continued to mate after 3 and 6 hours. cc124 and cc125 cells, however, did appear to be affected by the drug. After being suspended in the solution under constant illumination for three hours, no cells appeared to agglutinate and there was no cell wall loss. When the gametic cc124 and R3+ cells treated with folimycin were mixed together, aggregates of cells were observed which indicates that there could have been some agglutination; however, after a few seconds the cells dissipated from one another. In both of these experiments, mating/agglutination ability returned after six hours. We believe that the cells are unaffected after a certain time period because folimycin is extremely sensitive to light. Because C. reinhardtii needs light to become gametic, the overexposure probably stopped this drug from working after a certain length of time.

Once it had been determined that DMSO did not effect the cells; only NFHSM was used as a positive control solution. To account for folimycin's sensitivity to light, a second folimycin experiment was performed to assess agglutination after only thirty minutes of cell suspension. Thirty minutes, however, was not enough time for cc124 and cc125 cells to become gametic. As a result, mating was observed after 2 and 5 hours (Table 9). When gametic R3+ and NO- folimycin treated cells were mixed, there was significantly less mating and agglutination occurring among the cells as compared to the positive control (non-treated cells). Some agglutination occurred when the gametic cc124 and cc125 folimycin treated cells were mixed but the amount was significantly less when compared to the untreated cc124 and cc125 cells. As observed in the previous experiment, when folimycin treated cc124 was mixed with R3+, there was very little mating. However, when the folimycin treated cc125 cells were mixed with the folimycin t reated NO- cells there was mating and agglutination occurring. Because mating did occur with the folimycin treated NO- and R3+ cells, the folimycin drug appears to have some sort of effect on the cc124 cells only. At this point, additional experiments were necessary to try to determine more accurately if the drug was having a specific effect on the cc124 cells.

Thus, the third time this experiment was performed, cells were quantified using a hemacytometer in order to have a more accurate account of the drug's effects on the cells (Table 10). After thirty minutes, the cc124 and cc125 cells were mixed and agglutination was assessed. The number of mating pairs of folimycin treated cells was not much less than the number of mating untreated cells (e.g.15-8, 20-11, etc.). However, after 2.5 hours under constant illumination, thenumber of folimycin treated mating pairs was significantly less than the number of mating pairs for the untreated cells (e.g. 15-25, 12-30, etc.). The biggest difference was recorded after 3.5 hours. The untreated cells had 50+ "agglutinating clumps" in each of the four field views observed; however, the treated cells had a noticeably lower number of "clumps."

Table 10. Mating observations for treated and untreated cc124, cc125, NO- and R3+ cells. Four different fields were observed at 40X and the number of mating pairs were counted after 30 minutes, 2.5, 3.5, 5.5, and 7 hour.



The number of mating pairs for the NO- and R3+ cells were approximately the same in all experiments indicating that the folimycin has no real effect on these strains of cells. R3+ and NO-cells, however, were engineered to be efficient maters and are not very similar to wildtype GA1 cells. Therefore, an additional experiment was performed in order to determine if folimycin was having a specific effect on the cc124 cells. cc124 cells were mated with the R3+ cells , which we knew could mate based on the results from previous mating combinations. 5 fields at 40 X were observed after 1.5 hours and mating pairs were counted for each mating combination (Table 11). The folimycin treated cc124 and cc125 cells had few pairs of mating cells as compared to the untreated cells. One could assume, based on the significant difference in mating pairs, that the folimycin was having an effect on the mating between cc124 and cc125. Again, the treated cc124 and R3+ cells also had approximately the same number of mating pairs as the non-treated cells. Although the drug appeared to have some effect on the cells, it did not appear that folimycin could chemically mimic the phenotype of GA1 cells.

Table 11. Mating observations for treated and untreated cc124, cc125, NO- and R3+ cells. Five different fields were observed at 40X and the number of mating pairs were counted after one hour and 30 minutes.

A last attempt using the folimycin to chemically mimic the GA1 phenotype was made by varying the concentration of folimycin in the solutions. No differences in mating among any of the strains of cells were seen after 1.5 hours (Table 12). Therefore, we decided to abdandon the folimycin experiments as well.



folimycin

wt- strain

wt+ strain

Field#1 @40X

Field#2 @40X

Field#3 @40X

Field#4 @40X

Field#5 @40 X

-

NO-

R3+

15

20

17

11

13
.4 µl

NO-

R3+

13

20

17

11

13
.8 µl

NO-

R3+

12

16

18

11

9
-

cc124

cc125

10

12

11

13

8
.4 µl

cc124

cc125

8

7

9

6

5
.4 µl

cc124

R3+

10

6

8

12

9
-

cc124

R3+

12

11

8

10

13

Table 12. Mating observations for treated and untreated cc124, cc125, NO- and R3+ cells. Five different fields were observed at 40X and the number of mating pairs were counted after 1.5 hours.

At this point in our experimentation, more of the cDNA had been sequenced which revealed that GA1 is more similar to a plasma membrane H-ATPase. Therefore, our efforts to try and chemically mimic the GA1 phenotype were abandoned

III. Tetrad Analysis

To further characterize GA1 in terms of its chromosomal location and to determine whether or not mt+ strains of C.reinhardtii could have the GA1 phenotype, tetrad analysis was performed. The results from the tetrad analysis in this experiment indicate that we did not have tetrads because all four colonies for each "tetrad" were of the same mating type. If they were true tetrads, two of the strains should have been mt- and two should have been mt+. Because tetrad analysis is very time consuming, we decided to focus our extra efforts on subcloning the rest of the gene.

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