1

The Presence of Thioredoxin Gene

in Cyanopodoviruses

Presented By: Liron Schwartz 305022998

Supervisors: Prof. Debbie Lindell & Shay Kirzner

Date: 04/08/2013

Photograph was taken from Dekel-Bird et al., 2013.

2

Introduction

Marine cyanobacteria of the genera Prochlorochoccus and Synechococcus are highly

abundant organisms in oceanic waters1. Since they are oxygenic phototrophs and do

photosynthesis, they have a great contribution to the global primary productivity2. Viruses that

infect cyanobacteria (cyanophages) are the most abundant particles in marine ecosystems3,

therefore they have a strong impact on both host abundance3,4

and diversity5–7

.

These

bacteriophages belong to the Caudovirales order, in which a virion is composed of an

icosohedral head, that contains the double-stranded DNA viral genome, and a flexible tail7–12

.

These bacteriophages are divided into three families according to tail morphology: The

T7-like Podoviridae with short tails

13–15, the T4-like and TIM5-like Myoviridae with contractile

tails16

and a diverse cyanophage group Siphoviridae with long non-contractile tails17,18

. The

cyanopodoviruses and cyanomyoviruses genomes range from 42kb to 47kb and 161kb to

252kb, respectively13–15,19,20

. The cyanosiphoviruses genomes range from 30kb to 108kb18

.

The cyanopodoviruse genome is similar to that of T7 phage in both gene content and

genomic architecture14

. It's genome is organized into three functional clusters that contain

putative host take-over genes, DNA replication genes and phage particle formation genes13–

15,19.

Some cyanopodophages encode host-like genes which they have acquired from their

hosts through co-evolution14,16,20–22

. These genes participate in oxygenic photosynthesis,

metabolic pathways and stress-response14,23–26

. For example, photosystem II reaction center

gene (psbA), which takes part in the light reactions of photosynthesis23,25,26

; high light inducible

stress response gene (hli)27

; transaldolase (talC) and ribo-nucleotide reductase (nrd)25

. While

talC inhibits Calvin's cycle it activates the pentose-phosphate cycle which produces NADPH

and carbon substrates required for nucleotide and DNA synthesis28

. The genes nrd, hli and

psbA are situated in cluster II whereas talC is located at the end of the genome in cluster III14

.

Despite the different positions of these genes, all of them are expressed during infection and are

transcribed together with DNA replication genes24

. It has been hypothesized accordingly that

they function together to produce carbonic compounds and energy during photosynthesis

thereby improving phage fitness24

.

P60-like clade cyanopodoviruses can be divided into two sub clades by DNA

polymerase gene diversity: MPP-A (marine picocyanopodoviruse A) and MPP-B (marine

picocyanopodoviruse B)19,29,30

. There are some differences between the two groups: clade A

phages infect primarily Synechococcus and have no psbA, hli and talC genes whereas clade B

phages infect either Synechococcus or Prochlorochoccus and almost all of them encode psbA,

hli and talC gene14,16,23,26,29,31–34

. Moreover, the phages associated to clade B have greater

diversity than of clade A phages29

. Currently, fourteen marine cyanopodoviruses genomes are

known13–15,19,35,36

.

Recent unpublished work has showed that clade A phages are more virulent and take

over the host faster than clade B phages. Furthermore, clade A phages replicate faster and

create bigger plaques soon after the infection than clade B phages (unpublished results).

3

In this study we wanted to deepen our understanding of why clade A phages are more

virulent than clade B phages? Specifically, we wished to discover the gene (or genes) in clade

A phages that causes the noted difference in the virulence.

There are a few possible hypotheses. One possible hypothesis is that variation in

encoding psbA, hli and talC genes in each clade may lead to different replication rates.

Additionally, gene interactions can influence the replication rate and virulence.

Another possible hypothesis is that variation in encoding thioredoxin (trx) gene exists.

Trx (also called gp25) is a 294bp long15

, known in T7 phages. In the replication system of T7

phages, the protein encoded by trx forms a complex with gp5 which binds to the DNA

replication fork and to helicase37

. Trx increases the replication rate and the processivity of DNA

polymerase38–42

. While P60 and Syn5 encode trx13,15

TIP37 doesn't (unpublished results),

suggesting that some clade A cyanophages probably encode trx while clade B cyanophages

don't. The goal of this study is to check this hypothesis and examine a specific locus in the

genome of clade A and B phages for the presence of the trx gene.

Experimental Procedures

We focused on a specific locus in cluster II at the genome, from primase/helicase

(pri/hel) to DNA polymerase (Dpol) genes. Syn5 and TIP37 were used as positive and negative

controls of encoding trx, respectively. Trx presence was studied with lysates that had only

cyanopodoviruses (no contamination). Syn9 (a known cyanomyoviruse20

) was used as a

negative control for identification of cyanopodoviruses.

Four cyanopodoviruses from clade A (P-SSP9, TIP28, TIP33, TIP30) and eleven from

clade B (TIP45, TIP46, P-GSP1, P-SSP3, P-SSP2, P-SSP6, TIP39, TIP42, P-RSP1, TIP41 and

TIP67) were examined. The samples to be sequenced were chosen based on PCR results. Bands

from clade A lysates that supported the presence of trx (TIP28 in Trx-R, TIP28 in Pri-

Hel/Dpol, TIP33 in Trx-R) as well as unexpected bands from clade B lysates (TIP41 in Trx-L)

were sent to sequencing.

In order to perform sequencing of the desired fragments, we first needed to amplify the

DNA amount of each sample. We prepared several duplicates of each fragment (Table 1), with

the same PCR conditions. The amplified DNA fragments were cloned and then sent for

sequencing (Table 1). The primers used for sequencing were either SP6 or T7. We calculated

the coverage percentage of each homologues region from their homologues genes by dividing

the number of amino acids of the homologues region by the length of homologues proteins.

This index indicates the reliability of the results. Higher coverage percentage indicates stronger

similarity of the homologues region to the protein.

PCR primer design

The degenerate primers used for identification of cyanopodoviruses were known primers

complementary to the DNA polymerase gene: Dpol 143-F and 534-R10,30,32,43,44.

4

The identification of cyanomyoviruses was carried by g20 (a conserved T4-like viral

capsid assembly gene45

) primers46

.

Four different degenerate primers, Pri/Hel_F, Trx-R, Trx-F and Dpol_R, were designed

to explore the existence of trx. The melting temperatures of the primers were: 35.60C, 66.5

0C,

66.50C and 42.4

0C, respectively. These primers correspond to three locations along the

investigated locus: Pri/Hel Dpol, Trx-Right (Trx-R) and Trx-Left (Trx-L). Pri/Hel Dpol

segment contains the whole locus between primase/helicase and DNA polymerase. Trx-R

segment contains the right half of trx and Dpol and Trx-L segment contains the locus between

the left half of trx and pri/hel, as illustrated in Figure 1. Syn5 and TIP37 have different Pri/Hel

Dpol locus content15

(the information about TIP37 is derived from unpublished results). Clade

A and B phages are expected to have similar Pri/Hel Dpol content as Syn5 and TIP37,

respectively (Figure 1). The expected PCR fragments are presented in Table 2.

PCR conditions

PCRs were carried out in a total volume of 25 µl, including 2µM forward and reverse primers,

0.2mM dNTPs, 1X reaction buffer, 0.08 u/µl BIO-X-ACT DNA polymerase and 1 µl phage

lysate as template. All PCRs had the same cycling conditions with different annealing

temperatures (Table 3). The conditions included an initial 5 min denaturing step at 95°C

followed by 40 cycles of denaturation at 95°C for 45 sec, annealing at 35–500C for 0.75-1 min,

elongation at 70°C for 45 sec, and a final elongation step at 70°C for 10 min.

Gel electrophoresis

All tests were carried on 2% agarose gel. The gel consisted of TAE buffer that was also used as

a running buffer. A 100bp ladder was used for the PCR products runs.

In order to check plasmid insertion, it was cleaved by EcoR-I endonuclease, and

fragments were separated by length in gel electrophoresis. A 1kb ladder was used to distinguish

between samples containing an insert and samples with plasmid only. A closed empty plasmid

was used as a control. One ladder was used for several clones.

DNA extraction from the gel

PCR fragments were excised from the gel and purified with the MinElute gel extraction kit

(Qiagen). The extracted DNA concentration was measured by nano-drop analysis.

Cloning

The gel purified fragments were inserted into pCRII-TOPO (Invitrogen) cloning vectors with

an ampicillin resistance gene and a β-galactosidase marker. The plasmids were transformed

into E. coli DH5α for blue/white screening. This strain has deletions of β-galactosidase (lacZ)

gene, so colonies containing an empty plasmid will turn blue. Colonies containing a plasmid

with an insert will turn white since it disrupts the lacZ expression. Ampicillin was added to the

growth medium in order to prevent the growth of bacteria that didn't take up the plasmid during

5

B.

Pri/Hel DNA Polymerase

~0.5kb

the transformation. Finally, plasmids were extracted from the bacteria using the QIAprep

Miniprep Kit (Qiagen).

Comparative genomics

The sequences of the primers and plasmids were excluded from the sequencing results.

Sequences of the inserts were compared (at the nucleotide level) with a nucleotide collection

database using TBLASTX program (NCBI).

Figure 1. Illustration of the Pri/Hel Dpol locus in cluster A and B phages with the

designed primers. (A) Pri/Hel Dpol locus of clade A phages, based on Syn5 known locus;

(B) Pri/Hel Dpol locus of clade B phages, based on TIP37 known locus. Arrows represent

the primers and their positions on the genome. The gray primers confine the whole Pri/Hel

Dpol segment, whereas the gray-red couples define the Trx-L and Trx-R segments.

Table 1. Data on PCR repeats and clones.

Sequence region PCR repeats Number of clones Number of clones sent for sequencing

TIP28 Trx-R 5 2 2

TIP33 Trx-R 8 2 1

TIP28 Pri-Hel/Dpol 4 3 1

TIP41 Trx-L 6 5 1

The number of PCR repeats ranged from 4 to 8 and the number of clones from 2 to 5.

Only one or two clones were sent for sequencing as it was sufficient for our analysis.

Table 2. The expected PCR fragments of Pri-Hel/Dpol, Trx-L and Trx-R.

Ø Pri-Hel/Dpol Trx-L Trx-R

Clade A ~1kb ~250bp ~800bp

Clade B ~0.5kb / ? - -

Since there is no previous information on the Pri-Hel/Dpol segment of cluster B phages,

there are two possible options. If trx is present in the segment, we expect to see a ~0.5kb

band. If not, the band length depends on the existence of other genes. If there are no genes

between Pri/Hel and DNA polymerase then no bands are expected. Otherwise, band length

will be correlated to the size of the genes present.

~250bp ~800bp

Pri/Hel trx gp 26 DNA Polymerase

~3kbp

A.

6

Table 3. Primers annealing duration and temperature.

Primers Annealing temperatures Annealing

duration

Dpol 500C 45sec

g20 350C 1min

Pri/Hel Dpol 400C* 1min

Trx-R 500C 45sec

Trx-L 400C 1min

The annealing temperatures of the primers ranged from 350-50

0C and the annealing duration

ranged from 0.75-1min. *In order to increase the specificity of the Pri/Hel Dpol primers, a

few tests were performed in 450-50

0C annealing temperatures.

Results

The identification of cyanopodoviruses results indicated that P-SSP9 and P-RSP1 lysates had

cyanomyoviruses (bands appeared for g20 primers) and therefore these lysates were excluded from the next

tests. Samples TIP45, TIP46 and TIP67 in Trx-L fragment weren't analyzed in PCR. The gel

electrophoresis results of PCR products are presented in Table 4.

Gel electrophoresis results of TIP28 in the Trx-R segment, TIP33 in the Trx-R segment and TIP28

in Pri/Hel Dpol fragment are presented in Figures 2A, 4 and 6A, respectively.

Verification of incorporation of TIP28 in the Trx-R segment, TIP28 in Pri/Hel Dpol fragment and

TIP41 in Trx-L segment inserts is presented in Figures 2B, 6B and 7, respectively.

The lengths of the obtained sequences of inserts are presented in Table 5.

The sequencing results indicated that TIP28 in the Trx-R segment contained 3 homologues regions to

Syn5: DNA polymerase, gp26 and trx (Figure 3). The lengths of these proteins are: 583, 83 and 99 amino

acids respectively. TIP33 in the Trx-R segment contained 3 homologous regions to Syn5: gp5, gp6 and gp7

(Figure 5). The lengths of these proteins are: 114, 54 and 60 amino acids respectively. TIP28 in Pri/Hel

Dpol fragment showed no common homologues areas to known phages. TIP41 in Trx-L segment contained

homologous region to KBS-P-1A cyanophage: hypothetical protein containing 154 amino acids (Figure 8).

7

Table 4. The gel electrophoresis results of PCR products of Pri-Hel/Dpol, Trx-L and Trx-R

segments.

Ø Host Pri-Hel/Dpol Trx-L Trx-R

TIP28 CC9605 ~1kb ~300bp ~800bp

Syn5 WH8109 ~1kb ~250bp ~800bp

TIP33 WH8109 ~0.5kb (1/2) / - (1/2) - ~800bp (3/4) / - (1/4)

TIP30 WH8109 - - ~800bp (1/4) / - (3/4)

TIP37 WH8109 ~0.5kb - -

TIP45 WH8109 - -

TIP 46 RCC307 - -

P-GSP1 MED4 - - -

P-SSP3 MIT9312 - - -

P-SSP2 MIT9312 - - -

P-SSP6 MIT9515 - - -

TIP39 MIT9215 - - ~0.5kb (1/2) / - (1/2)

TIP42 MED4 - - -

TIP41 CC9605 - ~0.5kb ~0.5kb (1/2) / - (1/2)

TIP67 CC9605 - -

Clade A phages are labeled in red, clade B phages in blue. The host of each phage is presented.

'-' denotes that no bands were received and '/' separates between two conflicting experimental

results. In brackets is the ratio of number of presented result per all executed trials.

For all segments, the controls (Syn5 and TIP37) gave the expected bands (Table 2). Most of

type B phages didn't show bands related to trx presence. Some of clade A phages results

supported trx presence whereas others didn't.

Table 5. Sequencing results: lengths of inserts of the sequenced regions.

The primers used were either SP6 or T7. The lengths of inserts ranged from ~500bp to ~1kb.

Sequence region Primers of the clones Length of insert (bp)

TIP28 Trx-R

clone I-SP6 763

clone I-T7 924

clone II-SP6 931

clone II-T7 940

TIP33 Trx-R clone I-SP6 493

clone I-T7 604

TIP28 Pri Hel/Dpol clone I-SP6 947

TIP41 Trx-L clone I-SP6 668

8

Figure 2. Samples of TIP28 in Trx-R segment.

(A) Gel electrophoresis results of TIP28 with Trx-R primers. Almost all the samples gave

~800bp band, similar to Syn5. (B) Verification of incorporation of TIP28 insert to plasmid.

All samples had 4kb (plasmid) and ~1kb bands.

Figure 3. Homologues regions analyses of TIP28 in Trx-R

fragment to Syn5.

The homology regions with SP6 primers that were depicted: (A) DNA polymerase; (B)

gp26; with T7 primers: (C) trx. Bit score ranged from 74 to 203 and the identities score

A.

hom

B.

C.

9

ranged from 53% to 77%. The coverage percentage of the homologues regions of the

homologues genes were 20%, 94% and 50%, respectively.

Figure 4. Samples of TIP33 in Trx-R segment.

All samples had ~800bp intense band, similar to Syn5.

Figure 5. Homologues regions analyses of TIP33 in Trx-R fragment to Syn5.

The homology region with T7 primers that were depicted: (A) gp5; and with SP6 primers:

(B) gp5, gp6 and gp7. Bit score ranged from 137 to 162 and the identities score ranged

from 69% to 79%. The coverage percentage of the homologues regions of the homologues

genes were 76% and 37%, respectively.

A.

B.

10

Figure 6. Samples of TIP28 in Pri/Hel Dpol fragment.

(A) Gel electrophoresis results of TIP28 fragments generated by Pri/Hel Dpol primers.

Almost all samples gave ~1kb band, similar to Syn5. (B) Verification of incorporation of

TIP28 insert to plasmid. All samples had 4kb (plasmid) and ~1kb bands.

Figure 7. Verification of incorporation of TIP41 in Trx-L insert.

Most samples and the closed empty plasmid contained ~700bp insert.

Figure 8. Homologues regions analyses of TIP41 in Trx-L fragment to KBS-P-1A.

The homology region consisted of hypothetical protein. The coverage percentage of the

homologues region of the homologues gene was 60%.

11

Discussion

The PCR results (Table 4) exhibited similar bands to the expected ones (Table 2)

and support the hypothesis that clade A phages encode trx while clade B phages don't.

Nevertheless, there were some exceptional results: missing bands, variability in results and

appearance of unexpected bands. Lack of bands, for example in all clade B phages and

TIP30 in Pri-Hel/Dpol segment (Table 4), might have been caused by a mismatch of

primer sequence to the phage genome (in which trx might still be present) or indicates the

absence of trx in the phage genome.

Some samples gave varied results with different frequencies. In the case of TIP33 in Pri-

Hel/Dpol segment, since many samples in this fragment didn't show bands as expected, the

most likely reason is lack of proper annealing of primers to the fragments. In the case of

TIP33 and TIP30 in Trx-R fragment, when bands were received, they were similar to the

positive control (~800bp) corroborating trx presence. On the contrary, the analyses of

TIP41 and TIP39 in Trx-R fragment should be repeated since the results were not

conclusive. TIP41 in Trx-L fragment showed an unexpected band (~0.5kb), probably due

to low specificity of the primers (caused by low annealing temperature) which can result in

unspecific binding to irrelevant sequences.

There are many possible sources for error such as human inaccuracies in pipetting

and handling samples, measurement errors, drift of samples during gel loading and more.

The sequencing results indicate that TIP28 had homologous regions to trx with

high identities score (>20%, see Figure 3), implying similarity of trx function. The

coverage percentage of the homologues region was 50% (Figure 3C) namely

approximately fifty percent of TIP28 encoded protein amino-acids sequence is similar to

trx protein. In Pri-Hel/Dpol segment no homologous regions were found, probably

because a nearby band was mistakenly excised from the gel and cloned. TIP33 had

homologous regions to gp5, gp6 and gp7 with high identities score (Figure 5). These genes

are preliminary host-take over genes positioned at cluster I at the genome of Syn515

. Thus,

they might also influence and contribute to high virulence. Gp5 is known to form a

complex with trx during replication in T7 phages37

hence, its presence supports the

assumption that trx mechanism does exist in clade A phages. Trx was not found in TIP41

in Trx-L segment (Figure 8).

In this work, trx presence was examined only in the specific locus of Pri/Hel Dpol,

though potentially it may be found at other loci in the genome. Our conclusions should be

critically assessed since the absence of trx from the locus does not necessarily rule out its

presence in the whole genome.

In addition to experimental sequencing analysis, we used two-way BLAST

program to compare between trx sequence and known clade B phages genomes (P-SSP11,

P-SSP10, P-HP1, P-GSP1, P-SSP7,P-SSP3, P-SSP2 and P-RSP5). No homologues regions

were found in any of the examined genomes. Moreover, the same method was used on

clade A phage strain P-SSP9 and also no homology was received.

12

To conclude, our findings corroborate the assumption that some of cluster A phages

encode trx while all of cluster B phages don't. Furthermore, we discovered the existence of

several other genes – gp5, gp6 and gp7 – potentially also related to clade A phages

increased virulence.

As mentioned above, some of our results were equivocal, thus further analyses are

recommended. The most accurate and easiest way to know whether trx is present in a

phage genome is to sequence the genome and use two-way BLAST analysis. However, this

procedure is cumbersome and laborious since it requires finding specific primers for each

phage. Another possible method is to perform PCR procedures with primers other than the

ones used in this assay. The chosen primers should have a more extensive region of Pri-

Hel/Dpol segment and preferably more appropriate annealing temperatures in order to

increase the specificity. Because gp5 has a role in the trx mechanism, a more extensive

research on this gene is advocated. To fully understand the difference in virulence between

clade A and B cyanopodophages, other hypotheses, such as the presence of psbA, hli and

talc genes, should be studied and explored.

13

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41. Wuite, G. J. L., Smith, S. B., Young, M., Keller, D. & Bustamante, C. Single-molecule studies of the effect of template tension on T7 DNA polymerase activity. Nature 404, 103–106 (2000).

42. Etson, C. M., Hamdan, S. M., Richardson, C. C. & Oijen, A. M. van. Thioredoxin suppresses microscopic hopping of T7 DNA polymerase on duplex DNA. Proc. Natl. Acad. Sci. 107, 1900–1905 (2010).

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Supplementary Information

The discovered sequences of the different segments are presented below.

TIP28 in Trx-R segment with SP6 primer, clone I:

NNNNNNNNNNNNNNNNNNNNNTTGGTACCGANCNNGNNTCCNCTAGTAACGG

CCGCCNGTGTGCTGGAATTCGCCCTTCTCGAGGGAGTGGCGACCGTAAAGGTT

GGCGGGCATGTTGGCTGGTCGGGAGCGGAAGTCACGATCCAGCAGGTCGGTG

AAGAACAACCGCGAGAGGATGAGAGTGTCATAGGTCTGTCCTTTGAACTCCCA

GTTCGGATGGATCTTCTTGATGGCCTGGTAGTCATAACCAATAATGTTGTGACC

CCAGACCTCGTCTGCATCCTTCAGCATCTTTAGGCCCTTCTTGTAATCAGAAGG

ACCAAAGCGGTACTTAGTACCGTCGTCGATGTTGATCAGAACAATGCAGTGGA

TCTTGGTGAGATCCTGAAGGAGACCATCGGTCTCAATGTCAAACGCTAGACGC

ATTGAATAGCTTGGGTTTGATACGTCCGAAACCAGAACGGATCTCCAGCACGG

CATAGCCGGCGTCATGGAGGTGGTCGAAGATGTCAACCTGGCGGAATGCCCGG

ATGGCGGAGACTTTCTCCTTCTTCTTCCAGCTAGGTTTCTTGTATCGGACGAGG

TGGATATCGGAGGGAAGCTCAGCGTCGAGCTTCTTAAGCTTTTCGAGAGAGGT

ACTCTCGATGTGAATNANCACNGGATCCTTCATCGATTACGGAANTAGTNNNT

ATGGCGGTTGATAGTTGNNAGGAGATTCACGATGTTGNNCNNATNCNNTGNNN

ACCNNCGANGNNGNNNNNTN

TIP28 in Trx-R segment with T7 primer, clone I:

NNNNNNNCNNNNNNNNNTGCTCGNNCGGNNGNCNGTGTGATGGATATCTGCA

GAATTCGCCCTTAAGGAGAATCNTTGGGCACTTGTTGAGGCTTACCNGCTGGA

TCTCTTCCCGACCTTCCTGATCGTGGATGACCACGGTGAGGAGATCGACCGCCT

CGTTGGTGGTCAACGTATTCGCGACAACATCGTGAATCTCCTTACAACTATCAA

CCGCCATAACGACTACTTCCGTAATCGATGAAGGATCCTGTGATTATTCACATC

GAGAGTACCTCTCTCGAAAAGCTTAAGAAGCTCGACGCTGAGCTTCCCTCCGA

TATCCACCTCGTCCGATACAAGAAACCTAGCTGGAAGAAGAAGGAGAAAGTC

TCCGCCATCCGGGCATTCCGCCAGGTTGACATCTTCGACCACCTCCATGACGCC

GGCTATGCCGTGCTGGAGATCCGTTCTGGTTTCGGACGTATCAAACCCAAGCT

ATTCAATGCGTCTAGCGTTTGACATTGAGACCGATGGTCTCCTTCAGGATCTCA

CCAAGATCCACTGCATTGTTCTGATCAACATCGACGACGGTACTAAGTACCGC

TTTGGTCCTTCTGATTACAAGAAGGGCCTAAAGATGCTGAAGGATGCAGACGA

GGTCTGGGGTCACAACATTATTGGTTATGACTACCAGGCCATCAAGAAGATCC

ATCCGAACTGGGAGTTCAAAGGACAGACCTATGACACTCTCATCCTCTCGCGG

TTGTTCTTCACCGACCTGCTGGATCGTGACTTCCGCTCCCGACCAGCCAACATG

CCCGCCAACCTTTACNGTCGCCACTCCCTCGAGAAGGGCGAATTCCAGCACAC

TGGCGGCCGTTACTAGTGGATCCGAGCTCGGTACCAAGCTTGATGCATAGCTT

GAGTATTCTATAGTGTNNN

17

TIP28 in Trx-R segment with SP6 primer, clone II:

NNNNNNNNNNGNNNNNNNTTGGTACCGANCTNGGNTCCNCTAGTAACGGCCG

CCNGTGTGCTGGAATTCGCCCTTTTCGAGGGTGTGGCGACCGTAAAGGTTGGC

GGGCATGTTGGCTGGTCGGGAGCGGAAGTCACGATCCAGCAGGTCGGTGAAG

AACAACCGCGAGAGGATGAGAGTGTCATAGGTCTGTCCTTTGAACTCCCAGTT

CGGATGGATCTTCTTGATGGTCTGGTAGTCATAACCAATAATGTTGTGACCCCA

GACCTCGTCTGCATCCTTCAGCATCTTTAGGCCCTTCTTGTAATCAGAAGGACC

AAAGCGGTACTTAGTACCGTCGTCGATGTTGATCAGAACAATGCAGTGGATCT

TGGTGAGATCCTGAAGGAGACCATCGGTCTCAATGTCAAACGCTAGACGCATT

GAATAGTTTGGGTTTGATACGTCCGAAACCAGAACGGATCTCCAGCACGGCAT

AGCCGGCGTCATGGAGGTGGTCGAAGATGTCAACCTGGCGGAATGCCCGGAT

GGCGGAGACTTTCTCCTTCTTCTTCCAGCTAGGTTTCTTGTATCGGACGAGGTG

GATATCGGAGGGAAGCTCAGCGTCGAGCTTCTTAAGCTTTTCNAGAGAGGTAC

TCTCGATGTGAATAATCACAGGATCCTTCATCGATTACGGAAGTAGTCGTTATG

GCGGTTGATAGTTGTAAGGAGATTCACGATGTTGTCGCGAATACGTTGACCAC

CAACGAGGCGGTCGATCTCCTCACCCGTGGTCATCCNCGATCAGGAAGGTCGG

GAANNAGATCCNGCTGGTAAGCCTCNACAAGTGCCCAATGATNCTCCTTNAGG

GCGAATTCTGCAGANATCCATCACACTGGCGGCCGNTNNNGCATGCATCNNAG

ANGGGCCCNATTNNCCCTATAGTGAGTCN

TIP28 in Trx-R segment with T7 primer, clone II:

NNNNNNNNGGNNCNNNNNNNNTGCTCGNNCGGCNGNCNGTGTGATGGATATC

TGCAGAATTCGCCCTTAAGGAGAATCNTTGGGCACTTGTTGAGGCTTACCAGC

TGGATCTCTTCCCGACCTTCCTGATCGTGGATGACCACGGTGAGGAGATCGAC

CGCCTCGTTGGTGGTCAACGTATTCGCGACAACATCGTGAATCTCCTTACAACT

ATCAACCGCCATAACGACTACTTCCGTAATCGATGAAGGATCCTGTGATTATTC

ACATCGAGAGTACCTCTCTCGAAAAGCTTAAGAAGCTCGACGCTGAGCTTCCC

TCCGATATCCACCTCGTCCGATACAAGAAACCTAGCTGGAAGAAGAAGGAGA

AAGTCTCCGCCATCCGGGCATTCCGCCAGGTTGACATCTTCGACCACCTCCATG

ACGCCGGCTATGCCGTGCTGGAGATCCGTTCTGGTTTCGGACGTATCAAACCC

AAACTATTCAATGCGTCTAGCGTTTGACATTGAGACCGATGGTCTCCTTCAGGA

TCTCACCAAGATCCACTGCATTGTTCTGATCAACATCGACGACGGTACTAAGT

ACCGCTTTGGTCCTTCTGATTACAAGAAGGGCCTAAAGATGCTGAAGGATGCA

GACGAGGTCTGGGGTCACAACATTATTGGTTATGACTACCAGACCATCAAGAA

GATCCATCCGAACTGGGAGTTCAAAGGACAGACCTATGACACTCTCATCCTCT

CGCGGTTGTTCTTCACCGACCTGCTGGATCGTGACTTCCGCTCCCGACCAGCCN

ACATGCCCGCCNNCCTTTACGGNCGCCACACCCTCGAAAAGGGCGAATTCCAG

CACACTGGCGGCCGTTACTAGTGGATCCGAGCTCGGTNNCNAGCTTGATGCAT

AGCTTGNGTATTCTATAGTGNCACCNNAATAGCTTN

18

TIP33 in Trx-R segment with T7 primer

NNNNNNNNNNNNNNNNNNNNNNNNGNTNGNNNGGGNNNNAGNNGTGATGGA

TATCTGCAGAATTCGCCCTTAAGGAGAACCNTAGGGCATCATGAACACCTGGG

AATNCTGGAACACCACCACACTNGNNNCCNACNACCTGATCGAGTTCGACTGN

GAAGGGNTTGAAGAGGCAGCCACCCTCCTCTCTNAACAGACAGACATCGATCC

AACTGAATGGGAGCTCTTCTNAATCAACGGCAACATCGTCTGACCCACCCACC

ACAGTTCACCATCACGCCCCGGCCAACGCTGGGGTTTTTTCATGCGAGCTCATA

GCGAGCTCTTGCCCAGCCCTCCCAATGTAAAGACAATGTGAAGAGTGGCACAA

AAGGGTTGACCTACCCATCCAGCCCGTATACCTTAGGNACATCGGANGCAAAC

ACAGCACTCCGAACNNACAACTGAAGAGGGNNGGACCACCCACCAAGNCCAG

CTAAACTGCTNAAGCTGCNGACCGNNNNNNNGATAGAGCTGACCTTNCNNNT

NANGGAAAAGANGGGAGCCTGAGNNNATNACTAGNCAGTGGATCGNGGGNN

GAACCNNCACTNNGGCCTTGNGANNN

TIP33 in Trx-R segment with SP6 primer

NNNNNNNNNNNNNNNNNNNNNCTTGGTACCGAGCTCGGNNCCNCTAGTAACG

GCCGCCAGTGTGCTGGAATTCGCCCTTCTCTAAGGTGTGGCGGGGACTGTTGA

GTAACGCATGATCAGATGGAAGGGAAGGAGTGAGTAACCTTGGTGACATCGT

AACCGAGCTGCTGCAGGGATGCAACTGCTTGGTGTGCAGAGGTGGCCATTACC

GGCATCTCGTGGCTGGTGTAGTTCCGGTCACGGTAGGTGATGCGGTAGGTGCG

GATCTGATTGAAATTCATGGCGTGATGATGGTGATCAGTTGTCAGCTTCCCAGC

GGGCCACTGCTGCAGCGTAAGCATTAGCGTGATCCATCTGGGCTTGGAGGCGA

GCGCGGCGTGCAGCAGGGCTGAGTGACCGGCGCTTGGAGTGCAGGCTGCTGTA

TTGCTTGCCTGCTTTGCGTCCGTTGGCCTTGATCATTTGAAGAGGTGGGGATGG

GTTTGAGGNNNNNNNN

19

TIP28 in Pri-hel/dpol segment with SP6 primer

NNNNNNNNNNNNNNNNNNNNNNNNNNNNTTGNNNNNNNNCTCGGNNCCCTN

GTAACGGCCGCCAGTGTGCTGGAATTCGCCCTTCTCGAGGGTGTGGCGACAAC

ACCAGCTGAGTTCAGCAGATGCAGAGTCTTTTTAAAGTAGCCACTATCAGCAT

CAGAAGCAAACTCACTTCCGATATCTACATTCGTAAACGTAAGTTTATTTACGA

CTCCGACAAGCGTTGAGCCGCCAGTGAAGTGCAACTTAACGCCCGTACCGCAG

GCTTTGTTAGTCTTAGCGTGAATAACTAAATTAGAGATAGCGTAAGCCTGATT

GGGGTTAGGCGTGCCATCGTTATACAGGAGTCTAAGGAAGTCATCACCCTCGC

TAGCCGGATCAAATACGATCCGAGAAGCCTGGCCATCCCCATACATGTGGAGA

CCTTTGTTTTCAACAGTGATCGTCGAGGTGACCAGATAGGTACCTGCAGGAAA

GTAGACGCTACCGCCAAGCTGCAAAGCATTGTTGATAGCAGTAGTGTCATCGG

TTGAGCCGTTTCCTACAGCACCTAGATCTCTAACGTTAGTGTAGCCACTAGCGC

CTTCACCACTACCGCCACACCCTAGAAAAGGGCGAATTCTGCAGATATCCATC

ACACTGGCGGCCGCTCGAGCATGCATCTAGAGGGCCCAATTCGCCCTATAGTG

AGTCGTATTACAATTCACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAAC

CCTGGCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGG

CGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCT

GAATGGCGAATGGACGCGCCCTGTANCGGCGCATTAAGCGCGGCGGGTGTGG

TGGTNACGCGCANCGNNGNCCNNTNNACTTGNCNGCGCCCTANCGC

TIP41 in Trx-L segment with primer T7

NTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTC

AGTGAGCGAGGAAGCGGAAGAGCGCCCAATACGCAAACCGCCTCTCCCCGCGC

GTTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGG

GCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGG

CTTTACACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACA

ATTTCACACAGGAAACAGCTATGACCATGATTACGCCAAGCTATTTAGGTGACA

CTATAGAATACTCAAGCTATGCATCAAGCTTGGTACCGAGCTCGGATCCACTAG

TAACGGCCGCCAGTGTGCTGGAATTCGCCCTTGCGCTGTGGTTTTCTTTTACTTT

TTCTAGGAAACCTTTGTTTCCTTCTTTTTTGTTAGCTTGCTCCATGTTTGTTTCAT

GATTGGTTTCATTATTGTGACCAGCCATTTAAATAAAGACGTAGCAGTTAGGGT

GGCGGCAACAGAAATAGTTGCTGTTGTAGCTGCTGTAGTCATAATAGTAGTTGT

TGGCATTGGTATTTCAATGTCCGTAAATGGAATCTCTACTATTTGAGCTTCAGGT

GGTAAATCTATATTGGGTTGTGCAGTCTGTTTTGTAGAAGTAGGTTTTTGTTCCT

CTGGGGGAGGACCATCTCCAACATTAATACCTTTAATACCTGGTGGTGGCTGTA

GTGTATTTGGTGGTACTACAAGCGGTTTGTAATTAGGTAAATCTGCCCGAGGCA

CCTCTAATACCGGAATTGGTAAATTAGGTGCATCAGGCAAAGATATGTAAGGTA

TTAAAGGAAAACCACCCTGCAAGGGCGAATTCTGCAGATATCCATCACACNGGC

GGCCG

20